Importation of Semen from Argentina
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IMPORT RISK ANALYSIS REPORT ON THE IMPORTATION OF BOVINE SEMEN AND EMBRYOS FROM ARGENTINA AND BRAZIL INTO AUSTRALIA PART 1: BOVINE SEMEN November 1999 Australian Quarantine and Inspection Service GPO Box 858 Canberra ACT 2601 AUSTRALIA TABLE OF CONTENTS EXECUTIVE SUMMARY 1 ABBREVIATIONS AND ACRONYMS 3 1. 4 INTRODUCTION 1.1 Scope of risk analysis 4 1.2 Current quarantine policy and practice 5 2. HAZARD IDENTIFICATION 6 3. RISK ASSESSMENT AND RISK MANAGEMENT 8 FOOT AND MOUTH DISEASE VIRUS VESICULAR STOMATITIS VIRUS BLUETONGUE VIRUS LEPTOSPIRA SPP RABIES VIRUS MYCOBACTERIUM PARATUBERCULOSIS BRUCELLA ABORTUS MYCOBACTERIUM BOVIS BOVINE LEUKEMIA VIRUS PASTEURELLA MULTOCIDA (SEROTYPES B:2 and E:2) Probability of disease transmission via infected semen BOVINE HERPESVIRUS-1 BOVINE PESTIVIRUS EPIZOOTIC HAEMORRHAGIC DISEASE VIRUS 4. ARGENTINA 4.1 5. 23 24 26 27 29 29 4.2. Occurrence of disease agents in Argentina Bovine leukemia virus 31 34 4.3 36 Summary of risk management measures BRAZIL 5.1 6. Argentina’s Veterinary Services 8 12 13 15 16 17 18 20 22 23 Brazil’s Veterinary Services 37 37 5.2. Occurrence of disease agents in Brazil 37 5.3 40 Summary of risk management measures REFERENCES 41 ATTACHMENT 1. 45 RISK ANALYSIS OF FOOT-AND-MOUTH DISEASE (FMD) IN THE ARGENTINE REPUBLIC 45 ii ATTACHMENT 2 OIE INTERNATIONAL ANIMAL HEALTH CODE ATTACHMENT 3. BOVINE SEMEN: OIE SANITARY CONTROL CONDITIONS 53 53 64 64 iii EXECUTIVE SUMMARY This import risk analysis (IRA) for the importation of bovine semen from Argentina and Brazil was undertaken in response to several enquiries from Australian cattle breeders interested in importing bovine genetic material from these countries. This document analyses the risks associated with importing bovine semen from Argentina and Brazil both of which present quite different animal health risks to the countries for which Australia has current bovine semen import requirements, viz the USA, Canada, New Zealand, Switzerland, Member States of the European Union (EU), Norway and New Caledonia. The diseases caused by the hazards identified in this IRA are quarantinable bovine diseases which could be imported with bovine semen and which could adversely affect the Australian livestock industry if introduced. Special consideration is given to Argentina’s recently acquired recognition, by the Office International des Epizooties (OIE), as a foot and mouth disease (FMD) free country where vaccination is practised. FMD was last reported in March 1994. Argentina has started to move towards becoming a country free from FMD where vaccination is not practised and has already prohibited FMD vaccination throughout the country. Progress in the eradication of FMD from Brazil is also considered. The risk assessment of diseases of concern includes consideration of epidemiological features affecting the likelihood of disease agents infecting or contaminating semen and the likelihood of the infected semen causing disease. In developing quarantine requirements, the disease status of Argentina and Brazil are considered. The following disease agents were identified as requiring risk management measures: foot and mouth disease virus, vesicular stomatitis virus, bluetongue virus, Leptospira spp, rabies virus, Mycobacterium paratuberculosis, Brucella abortus, Mycobacterium bovis, bovine leukemia virus, Pasteurella multocida (serotypes B:2 and E:2), bovine herpesvirus-1, bovine pestivirus, and epizootic haemorrhagic disease virus of deer. In many instances where disease freedom is not the sole option for managing the risk, the OIE International Animal Health Code (Code) recommendations were not considered to be adequate for managing the assessed risk according to Australia’s appropriate level of protection (ALOP). Modifications to some of the Code Articles are proposed and new risk measures formulated and proposed in some other cases. 1 The following risk management measures for List A diseases are proposed as requirements for the importation of bovine semen from affected countries or zones: : Foot-and-mouth disease The importation of semen from FMD affected countries and zones is prohibited as the current risk management options available do not provide adequate risk management measures according to Australia’s ALOP. However, for bovine semen from countries and zone officially free from FMD where vaccination is practised, it is proposed that donors are shown to be serologically negative to FMD antibodies before collection. Vesicular stomatitis Certification that during the 30 days immediately prior to collection that VS was not diagnosed within 15 km of any premises on which the donors resided. Bluetongue The recommendations of the OIE Code. The following risk management measures for List B diseases are proposed as requirements for the importation of bovine semen from affected countries or zones: Leptospirosis Antibiotics be added to semen during processing. Vampire bat rabies Certification that the donor showed no clinical signs of rabies. Johne’s disease The donor gave a negative result to an absorbed ELISA. Bovine brucellosis Donors originate from herds officially free from Br or from approved AI centres where the testing programme includes the buffered Brucella antigen and complement fixation tests. Bovine tuberculosis Either the donors originate from herds officially free from Tb, or the donors test negative to a prescribed series of intradermal tuberculin tests. Enzootic bovine leucosis The recommendations of the OIE Code. Haemorrhagic septicaemia Bovine semen from countries affected by HS is prohibited. Bovine herpesvirus Samples of the semen for export give a negative result to either a nucleic acid detection test or virus isolation by cell culture. The following risk management measures for other diseases are proposed as requirements for the importation of bovine semen from affected countries or zones: Bovine pestivirus Donors test negative to a virus identification technique during pre-entry isolation. Epizootic haemorrhagic disease virus Similar requirements to those adopted for BT. The recommendations outlined in the Code (Appendices 4.2.1.1. and 4.2.1.2.) are proposed as suitable standards for the management of artificial insemination centres and for the collection and processing of bovine semen. 2 ABBREVIATIONS AND ACRONYMS AGID AI ALOP AQIS AUSVETPLAN BHV-1 BLV Br BSE BT BTV BVD BVDV CFT Code CP DHS EBL EE EHD EHDV EITB ELISA FAO FMD FMDV HS IBR/IPV IRA JD MAARA MD NAMP NCP OIE PCR PI SENASA SNT SPS Agreement Tb USA VNT VS VSV WTO agar gel immunodiffusion (test) artificial insemination appropriate level of protection Australian Quarantine and Inspection Service Australian Veterinary Emergency Plan bovine herpesvirus-1 bovine leukemia virus bovine brucellosis bovine spongiform encephalopathy bluetongue bluetongue virus bovine viral diarrhoea bovine viral diarrhoea virus complement fixation test OIE International Animal Health Code 1999 cytopathic dihydrostreptomycin enzootic bovine leucosis equine encephalomyelitis epizootic haemorrhagic disease epizootic haemorrhagic disease virus enzyme-linked immunoelectrotransfer blot (assay) enzyme-linked immunosorbent assay Food and Agriculture Organisation of the United Nations foot and mouth disease foot and mouth disease virus haemorrhagic septicaemia infectious bovine rhinotracheitis/infectious pustular vulvovaginitis import risk analysis Johne’s disease Ministerio da Agricultura do Abastecimento e da Reforma Agraria (Brazil) mucosal disease National Arbovirus Monitoring Program non-cytopathic Office International des Epizooties polymerase chain reaction persistently infected Servicio Nacional de Sanidad y Calidad Agroalimentaria (Argentina) serum neutralisation test WTO Agreement on the Application of Sanitary and Phytosanitary Measures. bovine tuberculosis United States of America virus neutralisation test vesicular stomatitis vesicular stomatitis virus World Trade Organisation 3 1. INTRODUCTION 1.1 Scope of risk analysis This document analyses the quarantinable disease risks associated with importing frozen processed bovine semen from Argentina and Brazil into Australia. There are two main concerns associated with the widespread use of semen in artificial insemination - the dissemination of undesirable genetic traits and the transmission of exotic and other significant diseases. The former is not a quarantine concern. Both Argentina and Brazil have a number of diseases that are exotic to Australia as well as a number of enzootic diseases that are present at very low levels or are enzootic only in certain parts of Australia. Artificial insemination may transmit some of these diseases to susceptible females or even to their offspring via infected semen. This report: identifies the disease hazards which constitute a national quarantine risk when importing bovine semen; assesses the probability of bovine semen being infected with these disease agents; assesses the probability of infected bovine semen transmitting the disease agents causing disease; assesses the consequences if the diseases are introduced into Australia; identifies options for managing the risks of introducing disease into Australia with bovine semen, and proposes risk management options to be applied in respect of each disease hazard when importing semen from Argentina and Brazil. Factors that influence risk assessment include evaluation of the quality of veterinary services, surveillance programmes, and disease zoning systems. These factors can affect the probability of a pathogen infecting an animal population in the exporting country. Livestock breeders use artificial insemination to increase the rate of genetic gain of their livestock and to improve animal productivity on their farms. Several million cows are artificially inseminated each year in both Argentina and Brazil. Both countries have bovine gene pools that are of interest to Australian producers and there is a growing demand for importing bovine semen from Argentina and Brazil. Argentina and Brazil currently export bovine semen to other South American countries. The numbers of straws of semen and embryos exported from Argentina from 1995 to 1997 are given in Table 1. Table 1 Year 1995 1996 1997 Paraguay 1761 1846 1900 Brazil 3191 921 1722 Peru 227 200 0 Bolivia 45 0 0 Argentina and Brazil have imported bovine reproductive material from foot and mouth disease free zones and countries only. The numbers of straws of semen and embryos imported into Argentina are given in Table 2. (Note: Paraguay was recognised by OIE as a FMD free country where vaccination is practised at the same time as Argentina). 4 Table 2 Year 1995 1996 1997 1.2 Paraguay 0 11 0 Chile 0 17 220 Uruguay 400 2 716 USA 22 28 42 Canada 0 19 61 Current quarantine policy and practice The Quarantine Act (1908) provides for the Governor-General to prohibit, by proclamation, the importation of goods, if the importation of those goods into Australia is likely to introduce any disease or pest. The Quarantine Proclamation 1998 Section 27 lists animal semen, embryos or ova as prohibited biological materials. Section 35 defines animal reproductive material as part of an animal from which another animal can be reproduced, and includes semen, ova or an embryo. Section 28 (1) prohibits the introduction or importation of prohibited biological materials and Section 38 (1) prohibits the importation of animal parts into Australia, unless the Director of Quarantine has granted a permit to import as set out in Sections 28 (3) and 38 (4). Section 70 specifies that the Director of Quarantine, in deciding whether to grant a permit for the importation of semen, must consider: the quarantine risk and other relevant matters, and whether the imposition of conditions would be necessary to limit the quarantine risk to a level that would be acceptably low. Section 70 (1) defines quarantine risk as: the likelihood that the importation will lead to the introduction, establishment or spread of a disease or a pest in Australia; the likelihood that any such introduction, establishment or spread of a disease or pest will result in harm being caused to human beings, animals, plants, other aspects of the environment or economic activities as a result of the introduction, establishment or spread of the disease or pest, and the likely extent of any such harm. Quarantine requirements currently exist for the importation into Australia of bovine semen from the USA, Canada, New Zealand, Switzerland, Member States of the European Union, New Caledonia and Norway. Semen for export must be collected at licensed or accredited semen collection centres and managed according to the Code or equivalent national standards. To minimise the risk of importing diseases Australia requires donor animals and/or their semen at these centres to undergo disease testing before export. As the animal health status of Argentina and Brazil differs markedly from countries from which Australia currently imports bovine semen, the development of conditions for these countries requires an IRA. 5 2. HAZARD IDENTIFICATION Table 3 lists the disease agents that could be transmitted in bovine semen. These disease agents are grouped according to the Code. Other disease agents listed are those that can cause communicable diseases identified as potentially hazardous, and are of importance in the international trade of animals and animal products. Disease agents (hazards) that are endemic in Australia and are the subject of official control programs or internal restrictions are identified as requiring risk assessment. Table 3 Those disease agents considered to be a hazard Hazard (disease agent) Susceptible Species Risk of being found in semen of infected donors? Australian Health Status Argentinian Health Status Brazilian Health Status Risk Assessment needed? Enzootic with zone free from FMD with vaccination Sporadic Yes Disease agent causing OIE List A diseases Foot and mouth disease virus cloven hoofed animals Yes Not reported Officially free since 1871 Country free from FMD with vaccination Vesicular stomatitis virus cattle, horses, pigs, and humans cattle, pigs, sheep, goats cattle Yes - by extrinsic contamination Yes Not reported Last reported 1986 Not reported Free since 1923 Not reported Free since 1967 Not reported Not reported No Not reported Not reported since 1921 No cattle Yes Not reported Not reported Not reported No multiple species include humans cattle (nonclinical), sheep (clinical) Theoretically possible Not reported Not reported Not reported No Yes Enzootic region No virulent strains Disease suspected but presence not confirmed Serologic evidence only, no clinical disease Yes Enzootic No official control programs Enzootic No official control programs Not reported Lyssavrus in bats Enzootic Enzootic Disease suspected but presence not confirmed Enzootic outbreaks reported in cattle Enzootic Not reported since 1983 Yes – public health risk. All states require animals in AI centre to be free from leptospirosis No Rinderpest virus Mycoplasma mycoides subsp mycoides (cattle strain) Lumpy skin disease virus Rift Valley fever virus Bluetongue virus Yes Yes Disease agent causing OIE List B diseases Leptospira spp all vertebrates except birds Yes Coxiella burnettii mammals, birds, arthropods (mainly ticks) all warm blooded animals Yes Rabies virus Theoretically possible Mycobacterium paratuberculosis cattle, cattle strain may infect other ruminants Yes Enzootic in certain regions National control programs Brucella abortus cattle, humans cattle Yes Not reported Free since 1989 Low sporadic occurrence No official control Campylobacter fetus subsp fetus Yes Yes - to assess probability of introducing rabies in semen where rabies in cattle commonly reported. Yes – all states have regulatory requirements Enzootic Enzootic outbreaks reported in cattle Not reported since 1986 (cattle) and 1993 (sheep and goats) Enzootic Enzootic Enzootic No - no restrictions on sale of infected bulls; some states have no regulatory requirements for Yes 6 Hazard (disease agent) Susceptible Species Risk of being found in semen of infected donors? Australian Health Status Argentinian Health Status Brazilian Health Status programs Mycobacterium bovis cattle, deer, camels, humans, pigs cattle, sheep Yes Pasteurella multocida (Serotypes B:2 and E:2) cattle Yes extrinsic contamination possible Bovine herpesvirus1 Cattle Yes Tritrichomonas foetus Cattle Yes cattle (clinical) sheep and wildebeest Cattle Not reported Not likely Low sporadic occurrence but pathogenic BHV-1.1 not reported Low sporadic occurrence especially in northern parts. No official control programs in some states Exceptional occurrence No Bovine leukemia virus (BLV) Bovine malignant catarrhal fever virus Bovine spongiform encephalopathy prions Yes Sporadic - OIE classified free since 12/1997. Enzootic in certain regions Voluntary control programs only in dairy cattle Not reported Risk Assessment needed? campylobacteriosis in semen Yes Enzootic Enzootic Enzootic Enzootic Yes – the dairy industry in all states have EBL eradication programs. Not reported Yes Enzootic Reported sporadic but same expression for shipping fever Enzootic Enzootic Enzootic No - no restrictions on sale of infected bulls in some states; some states have no regulatory requirements for T foetus in semen Not reported Sporadic No Never reported Not reported Not reported No Enzootic - no pathogenic Type 2 recorded Serologic evidence only Enzootic Enzootic Yes Disease suspected but presence not confirmed Sporadic Not reported Yes Sporadic No Yes - to assess probability of introduction of virulent strains in semen Disease agent causing other diseases Bovine pestivirus Cattle, sheep, pigs Yes Epizootic haemorrhagic disease virus Cattle, deer, sheep Yes Cattle Yes Bovine lentivirus Low sporadic occurrence 7 3. RISK ASSESSMENT AND RISK MANAGEMENT Factors to consider in assessing the risk of introducing disease into Australia in frozen semen from other countries are: likelihood of a donor animal being infected with a disease agent; failure to detect the disease agent in a donor animal; likelihood of the disease agent being present in semen (intrinsic contamination) at collection; likelihood of extrinsic contamination of semen during collection, handling, and processing; failure to detect the disease agent in semen; failure to remove the contamination from semen with antibiotics during processing; likelihood of infected semen transmitting the disease agent to the recipient cow; likelihood of infected semen causing disease; likelihood of recipient cow spreading the disease; and consequences of the agent’s introduction and establishment of the disease in Australia. Whilst all of these factors need to be considered for each disease, not all of these factors are discussed for each disease. Some factors are not relevant to some agents, for example, it is not possible to remove intrinsic or extrinsic viral contamination from semen with antibiotics during semen processing. Options for management of risks in importing bovine semen into Australia can be influenced by the regulatory standards for AI centres and can be divided into four broad groups: country/zone certification of freedom from disease; testing of donors for freedom from disease agents; isolation of donors on entry to artificial insemination centres; and adoption of hygienic procedures during collection and processing of bovine semen. The following are discussed for each disease agent: the risks of introducing that agent in semen into Australia, the risks of that agent causing disease in Australia, the probable consequences of disease entry and establishment in Australia, and the risk management options. FOOT AND MOUTH DISEASE VIRUS The incubation period for foot and mouth disease (FMD) ranges from 2 to 14 days, with excretion of FMD virus (FMDV) up to four days before onset of clinical signs. High levels of FMDV can occur in all secretions and respiratory aerosols of infected animals. Urine and faeces can contain variable but usually low levels of the virus. The carrier state is an important epidemiological feature of FMD. After clinical recovery from FMD, up to 80% of ruminants may become carriers. In cattle, the pharyngeal and upper oesophageal regions are known sites for persistent infections which usually lasts for four to five months but have been reported to last for up to 42 months.27 Using nucleic acid tests, FMDV-specific genomic sequences can be detectable for at least 750 days after infection in spleen, lung, larynx, tonsils, pancreas, liver, oesophagus and white blood cells of cattle although no infective virus could be isolated from these organs by infectivity assays.30 Researchers believe that the virus does not pass into the salivary secretions of carriers. Although researchers have not been able to experimentally 8 demonstrate viral transmission from carrier animal, there is evidence that this does happen in the field. Sometimes, clinical disease occurs in susceptible animals a few months after the removal of carrier animals. Vaccinated animals may also become infected with the virus, despite having full protection against the disease, and could even become carriers of FMD. Antibodies to previous FMD infections can be detected in vaccinated animals for up to three years after vaccination. FMD vaccines are chemically inactivated tissue or organ culture derived preparations of the virus that have been blended with a suitable adjuvant. Before field use, vaccines are tested by using in vitro tests when inactivated and in vivo tests when finished to ensure that the product is non-infectious. Progress in FMD eradication in recent years has been largely due to significant advances in vaccine production technologies and the implementation of more efficient vaccine potency control programs. FMD vaccines now offer higher levels of protection of longer duration. The publication “OIE Manual of standards for diagnostic tests and vaccines” provides internationally approved standards required for the production of safe FMD vaccines. Probability of semen being infected The virus can be found in semen of transiently infected animals. FMDV has been detected in semen of experimentally infected animals for up to 56 days after infection. Infective virus can be isolated from wild buffalo semen and sheath wash.31 Vaccinated animals may become sub-clinically infected with FMD and there is also a probability that semen of vaccinated animals may contain FMDV. Recovered animals often become persistently infected and, as the virus can be found in semen of recovered animals after viral excretion from vesicles and foot lesions have ceased, it is likely that the virus can remain infective in seminal fluids and be present in semen during the carrier phase. Probability of disease transmission via infected semen Sexual transmission of FMDV has been suspected in some instances, especially between mature carrier Cape buffalo bulls and female cattle,31 but there are no field reports of FMD as a result of artificial insemination. Under experimental conditions infected semen has been shown to transmit the virus to other animals via insemination. The virus is capable of surviving indefinitely at ultra-low temperatures and should survive freezing in processed semen. Consequences of disease introduction into Australia Any incursion of FMD into Australia would have a serious impact. FMD could spread rapidly with significant loss of production, with flow-on effects to other sectors of the rural industry, gross domestic product and employment. All trade in ruminants, pigs and their products would cease until disease control and eradication restored international confidence in Australia’s FMD status. Estimation of risk It is highly unlikely that semen would be collected during the viraemic phase when the animals are clinically infected. However there is a moderate risk of persistently infected donors shedding FMDV in semen for about two months after infection. Also there is a low risk of vaccinated donors being subclinically infected with FMD and shedding FMDV in semen. Because of the severe consequences that an incursion of FMD may have in Australia and the risk of FMD being transmitted by artificial insemination, risk management measures are essential. 9 Risk management options and recommended measures The probability of importing FMDV in bovine semen would be reduced if the donor animals were not: incubating the virus, experiencing viraemia, persistently infected, or subclinically infected or seropositive to FMD while vaccinated against FMD, and shedding the virus in the semen. There are several serological tests for FMD. The recently developed standardised EITB assay, suited to the appropriate FMDV serotypes by selection of serologic probes, has proved to be an extremely sensitive and specific test for FMD and can detect antibodies associated with persistently infected animals under experimental conditions.2 Research suggests the test can also be used to differentiate antibodies from those arising from the vaccination response in cattle. However, this test has not performed as hoped when tested in the field. The EITB is routinely used as a valuable serological tool for FMD surveillance in several countries.3 FMDV infection can elicit antibodies specific for certain components of the FMDV. Vaccination elicits only antibodies to virus capsid proteins and the polymerase 3D while virus replication in cattle elicits additional antibodies directed against some non-structural (NS) proteins irrespective of prior vaccination or whether cattle developed clinical signs.28 While there is a promising assay for measuring antibody to the NS proteins Lb, 2C, 3A, 3D and 3ABC undergoing assessment for field work,29 the 3ABC indirect ELISA is preferred to the EITB for detecting carriers in individually vaccinated animals (Kitching P, pers comm) and these tests are more suited for use as a herd or group test rather than as an individual animal test.32 Consequently the simultaneous use of both tests for import/export testing, applied at maximum sensitivity, is proposed for export of susceptible animals from risk areas.48,49 Such risk areas include regions free from FMD within countries not officially free from FMD. Risk management options include: testing semen samples for FMDV - this may be possible with PCR tests, however none have been developed and assessed as yet; assessing the donor for freedom from FMDV clinically and serologically; vaccination of donors for protection against FMD; requiring that the donor originate from areas which have recorded no FMD outbreaks for a period long enough to eliminate the possibility of carriers or sub-clinically infected animals being present in the area and which excludes animals from FMD risk areas; or a combination of the above options. The risk of collecting infected semen from a vaccinated donor is negligible if the donor: tests negative to highly sensitive and specific tests such as standardised EITB assay suited to the appropriate FMDV serotypes, or both EITB and 3 ABC indirect ELISA, and resides in a zone where there has been no outbreak of FMD for at least 4 years. Thus measures to minimise the risk of introduction of FMDV via infected semen should require that the donors: are kept in an area free from FMD without vaccination, or are kept in an area free from FMD with vaccination for at least two years, and be seronegative to FMDV with tests of high sensitivity and specificity such as the enzyme10 linked immunoelectrotransfer blot (EITB) assay, suited to the appropriate FMDV serotypes; or both EITB and 3 ABC indirect ELISA prior to semen collection. The Code gives options for the importation of semen of domestic ruminants and pigs from FMD free countries or zones where vaccination is not practised (Article 2.1.1.8.); FMD free countries or zones where vaccination is practised (Article 2.1.1.9.), and FMD infected countries or zones (Article 2.1.1.10.). Although Article 2.1.1.10. provides the option of using semen from either a healthy vaccinated donor or a seronegative donor, there is a risk as donors can reside in areas where FMD outbreak have occurred within the previous 2 years, and donors may be false seronegative to the FMDV serotype being tested or be positive to a different FMDV serotype using the OIE recognised tests (blocking ELISA, VNT, and CFT). The EITB assay and 3 ABC indirect ELISA are not yet in the OIE Manual and the other recommended tests are not as sensitive or specific as these two tests. It is proposed that this option is unsuitable for Australia’s requirements. Article 2.1.1.9. does not entirely satisfy the proposed import requirements in that it lacks the proposed testing requirements. However the following modification of Article 2.1.1.9. is proposed (with modifications underlined),: The donor animals… b) were kept in a county or zone free from FMD with vaccination for at least 2 years or in a country free from FMD without vaccination for at least 1 year; c) If destined to an FMD free country or zone where vaccination is not practised EITHER i) have not been vaccinated and blood samples, drawn from each donor between 28 days and 60 days after final semen collection, showed a negative response to either the EITB assay using all appropriate serological probes for antibodies against FMD virus (if from a free country) or both the EITB and 3 ABC iELISA (if from a free zone). OR i) had been vaccinated at least twice, with the last vaccination not more than 12 and not less than 1 month prior to collection and blood samples, drawn from each donor between 28 days and 60 days after final semen collection, showed a negative response to either the EITB assay using all appropriate serological probes for antibodies against FMD virus (if from a free country) or both the EITB and 3 ABC iELISA (if from a free zone). Australia has not included reference to Article 2.1.1.8. in import conditions for bovine semen but has required that USA, Canada, New Zealand, Switzerland, Member States of the EU, New Caledonia and Norway meet the Code definition of a country free from FMD without vaccination (Article 2.1.1.2.). 11 VESICULAR STOMATITIS VIRUS Vesicular stomatitis (VS) is currently confined to the Americas, being endemic in Ossabaw Island in southeast United States, Central America, Venezuela, Colombia and Ecuador. Sporadic epizootics have occurred in other parts of USA and in neighbouring countries such as Canada, Brazil and Argentina. The epidemiology of vesicular stomatitis (VS) is not completely understood. Fomites as well as insect vectors may transmit the disease to susceptible animals. Sub-clinical infection is common. The incubation period is usually 2-3 days, with the infective period up to 21 days. The most recent outbreaks in USA were in 1986, 1995 and 1997-8. A post-epidemic survey in 1996 revealed some livestock, from holdings inspected during the 1995 outbreak and with no signs of VS, to be seropositive to VS. In 1995, the outbreak spread nearly 900 km in four months, mostly within the Rio Grande Valley. Spread appeared to be influenced by insect vectors and livestock movements. The only outbreak of VS known to have occurred outside the Americas was in Europe during the First World War following introduction of cavalry horses from USA. The disease appeared to have died out of its own accord. VSV seropositive animals have been detected in areas, such as Argentina, where sporadic outbreaks of VS have not occurred for a number of years. VS virus (VSV) is rapidly inactivated by common disinfectants and sunlight but can survive freezing. Handling cultures of VSV or infected animals can cause mild human infection with vesicles on the lips and tongue that regress quickly without complications. Infection can be mechanically transmitted to other people or animals. Over 37% of abattoir workers in a Colombian abattoir developed neutralising antibodies for VS.14 Probability of semen being infected Infection of semen as a result of disease in the animal has not been proven but there is a probability of extrinsic contamination of bovine semen in risk areas. Probability of disease transmission via infected semen Venereal transmission has not been proven, however it is probable for disease transmission to occur as a result of handling VSV contaminated material during artificial insemination. Consequences of disease introduction into Australia An incursion of VS into Australia could have a severe impact on the equine and dairy industry. It is not known whether Australia has suitable ecological conditions for the propagation of VSV. If these conditions exist, then it may be impossible to eradicate introduced VSV despite AUSVETPLAN strategies. However it is likely that the disease would be easily controlled and eradicated. Estimation of risk There is negligible risk of transmitting VS by artificial insemination, but there is a very low probability of transmitting VS as a result of handling contaminated equipment. Although sunlight and disinfectants readily inactivate VSV, the procedures during semen collection and processing are highly favourable for survival of the virus. Thus the virus is highly biohazardous and risk 12 management measures are necessary to ensure that semen, semen straws, and the transport containers are not contaminated with VS. Risk management options and recommended measures The continuing sporadic outbreaks in parts of the USA suggest that VSV cannot be eradicated from such areas. Thus such areas cannot be considered as being free even though no clinical, epidemiological or other evidence of VS occurs for many years between outbreaks. Risk management options are: the donors are kept in areas free from VSV; donors are kept on premises where VS has not been diagnosed during the 24 days immediately prior to collection; the donors give a negative response to tests for antibodies against VSV; or a combination of above options The standard interstate restriction that was imposed during the 1996 VS outbreak in USA was that “VS had not been diagnosed within 10 miles (15 km) of the premises of origin of these animals within the past 30 days.” This restriction was successful in minimising the spread of VS. The Code offers no recommendation concerning the importation of semen from free or infected countries. The Code (Article 2.1.2.2.) defines a VS free country as one when: VS is notifiable in the country, and no clinical, epidemiological or other evidence of VS has been found during the past two years. This definition does not recognise that sporadic outbreaks may recur more than two years after previous outbreaks, when ecological conditions become suitable for the propagation of VSV. The proposed risk management requirement is the same as that included in the conditions for bovine semen from the USA: VS was not diagnosed within 15 km of any premises where the donor was kept during the 30 days before the start of, and during, semen collection. BLUETONGUE VIRUS Bluetongue virus (BTV) occurs in many countries lying between 400 N and 350 S. BTV has a life-cycle alternating between vertebrate and invertebrate hosts and naturally infects domestic and wild ruminants. The disease is transmitted to vertebrate hosts by insect vectors in the genus Culicoides (gnats). Although most clinical disease occurs in sheep, cattle are the major vertebrate hosts of the virus. Incubation period is usually four to eight days and viraemia is mostly less than four weeks but may, in exceptional cases, be as long as eight weeks. 24 serotypes of BT are recognised and can be differentiated by serum neutralisation tests, although cross-reactions between some serotypes occur, and nucleic acid tests. Antibodies are usually first detected around one to two weeks after infection and can be detectable, with suitable serological tests, for a minimum of 60 days. 13 Probability of semen being infected BTV may sometimes be present in the semen of viraemic bulls. Probability of disease transmission via infected semen BT viraemia can occur in cows inseminated with infective semen. Infective semen does not appear to cause foetal death or developmental abnormalities. Evidence strongly suggests that calves produced from BT positive semen are BT antigen and antibody free. There have been no known cases of persistent infection and so there are no carrier states. There is a risk of introducing new strains of BT via infected semen if cows are inseminated in BT risk areas when vectors are active. Consequences of disease introduction into Australia Eight BT virus (BTV) serotypes (1, 3, 9, 15, 16, 20, 21 and 23) have been identified in Australia from insects or clinically healthy sentinel cattle but clinical BT disease has never been diagnosed in commercial sheep flocks or goats in Australia. If virulent BT were to be introduced into Australia, especially into areas with considerable Culicoides vector activity, it could spread and become extremely difficult to eradicate. It would have a severe impact on sheep production in the subtropical areas with interstate and international trade being affected. Information on the limited and seasonal distribution of BTV serotypes in Australia is already provided by NAMP’s extensive serological and vector collection data. This information would be used to help to reduce the socioeconomic consequences of the possible introduction of new strains of BTV. Estimation of risk There is a low probability that new strains of BTV could be introduced into Australia via BT infected semen. Risk management options and recommended measures The chapter on BT (Chapter 2.1.9.) in the Code is under review. The following certification options were recommended by the OIE Ad hoc Group on BT in September 1998 to minimise the risks of introducing BTV with imported bovine semen: EITHER the donors be kept in BTV free, or seasonally free, countries/zones for at least 60 days before commencement of, and during, semen collection; OR the donors be kept in a Culicoides-proof quarantine station for at least 60 days before commencement of, and during, semen collection; OR serum samples be collected from each donor at least 14 days before first semen collection, between 28 days and 60 days after final semen collection, and during semen collection period, at intervals of at least 28 days apart, if there is more than 60 days between the pre-collection and post-collection serum samples; and tested for bluetongue antibodies for each serotype of bluetongue known to occur in that country, with negative results in each case, OR blood samples were collected from each donor at the commencement of semen collection; 14 at the conclusion of semen collection; and either at least every 7 days during semen collection (for a virus isolation test) or every 28 days during semen collection (for a PCR). and subjected to a virus isolation test or nucleic acid detection test (polymerase chain reaction technology [PCR]) for bluetongue virus, with negative results. These options were outlined in the “Report of the meeting of the OIE Ad hoc Group on bluetongue” (Paris, 1-3 September 1998) and provide sound risk management strategies to prevent the incursion of virulent strains of BTV into Australia via bovine semen. LEPTOSPIRA SPP Leptospirosis is an important zoonotic disease with a worldwide distribution. There are some 26 serogroups containing over 230 serovars in the 8 species of pathogenic leptospires. It occurs in all species of domestic livestock as well as a number of wild animal species but not in birds. Vaccination of cattle can offer good protection against the serovars of leptospirosis included in the vaccine but serological testing does not differentiate between vaccinates and chronic carriers. Such tests do not have high sensitivity and consequently infected bulls excreting leptospires in semen can occasionally test negative. There is very little relationship between circulating antibodies and infected animals shedding leptospires. Antibiotic injections such as dihydrostreptomycin (DHS) are often used for clearing infections but efficacy may be a problem. Probability of semen being infected It is possible to recover the bacteria from organs such as kidney, seminal vesicle, epididymis, testes and semen of naturally infected bulls. Probability of disease transmission via infected semen Leptospires can survive in frozen semen especially when stored without antibiotics and can be transmitted by coitus or by artificial insemination. Consequences of disease introduction into Australia Two of the most common serovars, L borgpetersenii sv hardjo and L interrogans sv pomona, occur in cattle in Australia. Other serovars isolated from cattle include L interrogans svs australis,34 grippotyphosa35 and zanoni.36 Infections with some serovars such as L canicola and L icterohaemorrhagicae rarely occur in Australia but occur in cattle overseas. The only reports of L icterohaemorrhagicae infections in Australia are those acquired from rats and resulting in occasional outbreaks of canine leptospirosis. L canicola, though present in Australia, appears to be absent from the dog population and has never been detected in Australian cattle. Leptospirosis is endemic in Australia. Introduction of new strains may pose a public health risk and could cause loss of production in naive animals. 15 Estimation of risk There is a high risk that artificial insemination could be the means of transmitting leptospirosis to susceptible cattle when semen from infected bulls is used. Risk management options and recommended measures Antibiotics mixed with semen extenders are often used to minimise the risk of leptospires in semen. But there is growing concern about whether such measures are effective. Antibiotic resistance could be a problem with some leptospires but natural resistance of leptospires to antibiotics has not been reported (Faine S, pers comm). The standard protocol of adding antibiotic cocktails during processing of bovine semen is adequate to prevent the transmission of leptospirosis through AI.37 It is proposed that antibiotics be added to semen during processing as recommended in the Code Article 4.2.1.1.D. RABIES VIRUS There are several serotypes of rabies-related viruses. Within each serotype is a number of strains. Serotype 1 is the classical rabies virus strains isolated by Pasteur. A number of other strains have been identified within serotype 1 and these strains are maintained by specific reservoir hosts. In this section, emphasis is on the vampire bat strain, a serotype 1 strain. As the name suggests, this strain is maintained by the vampire bats of South America. This strain is not to be confused with the rabies related viruses of different serotypes isolated from frugivorous and insectivorous bats and bat lyssaviruses. Cattle are highly susceptible to rabies. Rabies in cattle rarely occurs except in Central and South America where it was estimated that several hundred thousand cattle died in outbreaks of rabies from rabid vampire bats each year during the early 1970’s. The losses have dropped considerably since following widespread use of rabies vaccine. Incubation period varies considerably. It is normally four to eight weeks, but can range from four days to over six months. This variability is related to the site of the bite, the virus strain and dose. Probability of semen being infected There are no reports of rabies being isolated from semen in livestock. However, rabies virus has been isolated from the testis of naturally infected vampire bats Desmodus rotundus in Brazil.18 The recent discovery that rabies can infect cornea and can cause transmission via corneal transplants has raised concerns about transmission of rabies.11 The virus can spread from the central nervous system via neural pathways and excrete in the saliva. It can disseminate to other tissues such as lungs, kidneys, heart, facial skin, and cornea. Thus there is a low probability of virus spread to the seminal vesicles and prostate glands, via neural pathways, and hence to the semen. The virus is comparatively fragile and does not retain infectivity for long away from the host. It is susceptible to most disinfectants and survives for only a few hours in dried saliva. However, bovine serum was found to be efficient in stabilising virus infectivity during repeated cycles of freezing and thawing.17 Furthermore, vaccines with recombinant virus have remained stable under field conditions of natural freezing and thawing.19 16 Probability of disease transmission via infected semen There are no reports of rabies isolated from semen nor have there been cases or suspicions of transmission of rabies via possible infected semen. Since rabies can be transmitted by corneal implants, there is a presumptive risk of transmission via semen. Consequences of disease introduction into Australia Australia is recognised as a rabies free country despite the presence of bat lyssavirus. Rabies has been reported in humans in Australia from overseas exposure but has not spread from index cases. If classical rabies became established in foxes or dogs in Australia the socio-economic consequences would be serious. However, as it is the vampire variant that is of concern in this IRA, and as there are no vampire bats in Australia, spread from the index case would be unlikely. Appropriate management of rabies cases generally requires vaccination of in-contact people and susceptible animals. Estimation of risk While the risk of cattle being infected by rabid dogs is very low, the risk of donor animals being infected with rabies is much higher in South American countries where vampire bat rabies occur. There is a very low risk of rabies infecting semen and of artificial insemination being the means of transmitting rabies to susceptible cattle. Cattle and humans are regarded as the end-host for rabies. Risk management options and recommended measures Risk management measures should be taken with bovine semen imported from South American countries where outbreaks of vampire rabies occur. Risk management should aim at ensuring that the donor animals were in a rabies free environment long enough to give the rabies virus adequate opportunity to manifest clinically. The Code offers no recommendation for risk management of bovine semen. However, it does recommend risk management options for: trade in live ruminants, pigs, and equines, that is, these animals showed no clinical sign of rabies on the day of shipment and they were kept for the six months prior to shipment in an establishment where no case of rabies was reported for at least 12 months prior to shipment (Code Article 3.1.5.6.). frozen semen in dogs, that is, the donor dogs showed no clinical signs of rabies during the 15 days following collection (Code Article 3.1.5.9.) The following certification would reduce the probability of importing rabies virus in bovine semen from Argentina and Brazil: The donor animal showed no clinical signs of rabies during, and for 15 days after, semen collection. Risk management is not necessary for other forms of rabies. MYCOBACTERIUM PARATUBERCULOSIS Probability of semen being infected The causative bacteria, Mycobacterium paratuberculosis, has been recovered from the testes, prostate, bulbourethral gland, seminal vesicles and semen from some infected bulls.40 17 Probability of disease transmission via infected semen Experiments indicate that transmission by artificial insemination with infected semen is unlikely but there are reports of congenital infections with bacteria being recovered from the infected foetus. Hence it is presumed that there is a very low risk of transmission of Johne’s disease (JD) with semen from bulls with clinical JD. Consequences of disease introduction into Australia JD is enzootic in certain regions in Australia. There is an official industry driven control program in Australia and JD is notifiable in all States and Territories. Australia is now divided into Free, Protected, Control and Residual Areas for Bovine JD. Western Australia is a Free Area and Queensland, Northern Territory and some of New South Wales are Protected Areas. A small proportion of New South Wales and all of Victoria and South Australia are Control Areas and Tasmania is a Residual Area. The zoning of JD has had adverse socio-economic impact on sheep farmers within the Control Areas. Introduction of JD into a Protected Area could result in its being declared a Control Area. Estimation of risk There is a very low risk of JD becoming established in a Protected Area if infected imported semen was inseminated in cows. However, the adverse socio-economic impact of its introduction justifies the need for quarantine measures for JD. Risk management options and recommended measures In order to manage the risk of importing JD infected semen, the donors need to be clinically, and tested, free from JD. The Code (Article 3.1.6.1.) provides no risk management options for semen but does recommend quarantine measures for cattle with JD. It is proposed that the donors test negative to JD with the absorbed ELISA after the first collection of semen but not more than 180 days after final collection. BRUCELLA ABORTUS Bovine brucellosis (Br) due to Brucella abortus is a highly contagious zoonotic disease characterised by late-term abortions and infertility in cattle. Although spread is usually by ingestion of infective material such as the placenta of aborted foetus, venereal transmission of Br also occurs. Br is found in most countries except where it has been successfully eradicated as a result of national regulatory programs. Countries where Br has been eradicated include Australia, Canada, Israel, Japan, Austria, Switzerland, Denmark, Finland, Norway, Sweden, and New Zealand. The USA has nearly eradicated Br. Unless vaccinated, animals infected with Br become infected for life. There is no treatment for Br. Vaccination may result in animals being seropositive to Br. Probability of semen being infected Brucella abortus localises in the testicles and seminal vesicles in infected bulls and can be shed in the semen. 18 Probability of disease transmission via infected semen The bacterium is capable of surviving freezing in processed semen. Artificial insemination can transmit Br and infect susceptible cows causing abortion in late pregnancy and infertility. Consequences of disease introduction into Australia Australia has successfully eradicated Br and has been officially free from this disease since 1989. Reintroduction of Br would put at risk the considerable investment into the national eradication of this disease. Br is a disease of major economic importance. As vaccination has been prohibited in Australia for a number of years, the cattle population is highly susceptible. It is expected that any incursion of Br would be detected and eradication measures instituted. Estimation of risk There is a high risk of transmitting brucellosis with semen from infected bulls. Risk management options and recommended measures Risk management is essential to prevent the reintroduction of B abortus into Australia via infected semen. Thus semen should only be collected from bulls free from this disease. Options to achieve this should depend on whether the area or herd where the donor resides is free from Br and not whether the individual donor is free from Br irrespective of herd status. The Code (Article 3.2.1.1.) (Bovine brucellosis) has definitions of country or part of the territory of a country free from Br, herd officially free from Br, and herd free from Br. Where semen is collected from donors in a country or part of the territory of a country free from Br, no further risk management should be necessary. Where semen is collected from donors kept in a herd free or officially free from Br, risk management is justifiable to ensure that the status is being maintained. Any donor bulls in such herds should have no clinical signs of Br and be serologically tested negative with recognised tests of high sensitivity and specificity to Br before semen is collected. Where semen is collected from donors not in areas officially recognised as being free from Br and not from Br free herds, then risk management should ensure that the donor(s) and all other animals residing on the establishment are clinically, serologically and antigenically free from Br. The options given in the Code (Article 3.2.1.4.) provide for risk measures for semen collected in or outside AI centres. Only semen collected from officially approved or accredited AI centres under the supervision of officially approved or accredited veterinarians is considered in this IRA. Furthermore, the herd or area status is not considered when collecting semen in AI centres. Article 3.2.1.4. does not provide the security required and the following risk management measures are proposed: Each donor animal EITHER were kept in a country or zone free from Br as defined by the OIE Code Article 3.2.1.1.) OR 19 immediately prior to collection were kept in a herd officially free from bovine brucellosis as defined by the Code Article 3.2.1.1 OR immediately prior to collection were kept in a herd free from bovine brucellosis as defined by the Code Article 3.2.1.1., and within 30 days prior to entering the AI centre, again during the pre-entry isolation period, and then at 6 monthly intervals , gave negative results to both the buffered Brucella plate agglutination test and the complement fixation test for bovine brucellosis, and prior to the export of semen gave a negative result to the buffered Brucella plate agglutination test for bovine brucellosis. MYCOBACTERIUM BOVIS Bovine tuberculosis (Tb), an infectious zoonotic disease caused by Mycobacterium bovis, is usually characterised by the formation of tubercles or nodular granulomas. Tb can infect a number of species of domestic and wild animals. Tb lesions may be found in most tissues but are more frequently observed in lymph nodes and lungs. Transmission of M bovis between animals is considered to generally be via aerosols, however other routes of infection are possible, including ingestion of infected material, via skin, teat canal, and the reproductive system. Infection is usually lifelong and treatment in cattle is uneconomic and unrewarding. Tb occurs worldwide. Although there are countries recognised as being officially free from Tb as defined by the Code (Article 3.2.3.1.), the definition requires 99.8% of the herds to be officially free from Tb. A range of tests are available for the detection of the Tb infection in cattle, including the intradermal skin test and blood-based laboratory tests. The Tb tests available are of lower sensitivity than most antibody detection tests for other diseases. Probability of semen being infected M bovis can infect semen intrinsically and extrinsically. Intrinsic infection can occur when mobile phagocytes containing viable M bovis facilitate the passage of the bacteria into semen. Extrinsic infection can occur in bulls with tuberculous lesions in the prepuce.24 Probability of disease transmission via infected semen M bovis can remain viable in frozen semen. Venereal transmission with semen intrinsically and extrinsically infected with M bovis has occurred. Consequences of disease introduction into Australia Australia is officially free from Tb. The reintroduction of Tb would put at risk the large industry and public investment in the national eradication of this disease. Control would be by quarantine, testing and slaughter and could be an expensive and time-consuming process. Estimation of risk 20 There is a moderate risk of transmitting M bovis with semen of infected bulls. Risk management measures are essential to prevent the reintroduction of Tb into Australia. Risk management options and recommended measures In order to manage the risk of importing Tb infected semen, the donors need to be clinically, and tested, free from Tb. Donors from countries which meet the definition of country or part of the territory of a country officially free from Tb as defined in the Code (Article 3.2.3.1.) should be considered as free from Tb and need no further risk management. For semen imported from countries/zones not officially free from Tb, the donors could be tested for Tb. The standard test for Tb is the single intradermal tuberculin test. Some false positives occur for which further testing is necessary. Of more concern is the low sensitivity (false negatives) with the single intradermal test. To improve sensitivity 2 tests are recommended. The Code (Article 3.2.3.7.) recommends 2 tests not less than 60 days apart. However, due to the desensitising effect that can last for up to 84 days, retesting is best done after 90 days. Donors residing in herds officially free from Tb as defined in the Code (Article 3.2.3.1.) should undergo the single tuberculin test annually with the rest of the herd to ensure the continuing absence of Tb. Such donors should be free from Tb and need no further risk management. Donors from herds not officially free from Tb are at the highest risk of being infected. Risk management is necessary to reduce the probability of collecting infected semen from such donors. To achieve this objective, the donor should be isolated from the herd at the premises of origin at least three months prior to preentry and undergo two tuberculin tests for Tb with negative results; and should undergo tuberculin tests for Tb with negative results at least 60 days after the last pre-entry test; at 6 monthly intervals; and before the release of semen for export if the last semen collection is more than 90 days after the previous test. The proposed certification requirement is: Each donor animal EITHER were kept in a country or zone officially free from bovine tuberculosis as defined by the OIE Code Article 3.2.3.1; OR immediately prior to, and during, collection, were kept in a herd officially free from bovine tuberculosis as defined by the OIE Code Article 3.2.3.1; OR gave negative results to intradermal tuberculin tests for Tb carried out: twice, at an interval of at least 90 days, during isolation from the herd at the premises of origin prior to entering the AI Centre; at least 60 days after the last pre-entry test; at 6 monthly intervals; and before the release of semen for export if the last semen collection is more than 90 days after the previous test. 21 BOVINE LEUKEMIA VIRUS Horizontal transmission of enzootic bovine leucosis (EBL) caused by bovine leukemia virus (BLV) may occur once the disease enters a herd. Successful transmission usually requires direct contact and prolonged exposure. Probability of semen being infected BLV infected leucocytes can leak into the semen of infected bulls. Probability of disease transmission via infected semen Results of transmission studies with semen and artificial insemination have been variable. Transmission may occur once infected leucocytes leak into the semen of infected bulls. In one case, 4714 cows, inseminated with semen from 30 leukotic bulls, gave birth to 1593 female calves 17 of which were positive. Later 22 cows became positive to EBL.39 The risk in this case was 3.6 infected calves per 1000 doses of semen (0.36%). Consequences of disease introduction into Australia The prevalence of EBL in Australia is very low. The Australian dairy industry has programmes of eradicating EBL from all dairy herds in Australia, under the State governments’ supervision. Western Australia (WA) is free from EBL and has a programme of milk testing to confirm their free status. A similar programme has been operating in Queensland (QLD) since 1983 and in New South Wales (NSW) since 1993. Victoria (VIC), South Australia (SA) and Tasmania (TAS) are also undertaking a programme of milk testing of all dairy herds to detect infected herds. All cattle in infected herds undergo serology testing (ELISA) and positive animals are culled. Most herds have been found to be free. EBL is now a quarantinable disease in NSW and farmers with BLV infected herds will receive 4 cents a litre less for their milk. Table 4 shows the number of dairy herds tested free from EBL at 31 March 1999. In some States not all herds have been tested and the difference between the number of free herds and the total number of herds in each state does not represent the number of infected herds. Table 4 EBL Free Herds Total Herds NSW 1536 1749 NT 0 0 QLD 1735 2026 SA 729 739 TAS 679 740 VIC 6481 8453 WA 455 455 AUST 11615 14162 For epidemiological reasons, the herd and within herd prevalence of EBL in beef cattle is very low in Australia. From 1 January 1998 to 31 March 1999 a total of 5838 serology tests for EBL in beef cattle were carried out at State veterinary laboratories, of which only 31 were positive. If BLV became established in WA, the State would lose its free status. If BLV became established in a herd previously free from EBL, there would be socio-economic effects on the farmer affected. Estimation of risk There is a low risk that BLV can be transmitted by semen from an infected donor. Risk management is necessary to prevent the introduction of BLV into EBL free herds in Australia. 22 Risk management options and recommended measures To minimise the risk of importing BLV infected semen, donors need to be clinically and serologically free from BLV. The Code (Article 3.2.4.4.) gives guidelines for bovine semen from donors tested negative for EBL and defines an EBL free herd (Code Article 3.2.4.2.). It is proposed the importation of bovine semen be permitted if it complies with OIE (Code Article 3.2.4.4.), that is: EITHER at the time of semen collection the donor was resident in an EBL free herd; and if less than 2 years of age, the donor came from a serologically negative "uterine" dam; OR the bull was subjected to diagnostic tests for EBL on blood samples on two occasions with negative results, the first test being carried out at least 30 days before and the second test at least 90 days after collection of the semen; AND the semen was collected, processed and stored in conformity with the provisions of the Code Appendices 4.2.1.1. and 4.2.1.2. PASTEURELLA MULTOCIDA (SEROTYPES B:2 and E:2) Haemorrhagic septicaemia (HS), a highly fatal disease of cattle and buffalo, is caused by either of two specific serotypes of Pasteurella multocida (Serotypes B:2 and E:2) which can be present in saliva, milk and urine in infected animals. Recovered animals may become carriers with the organisms being carried in the nasopharyngeal region, particularly in the tonsils and lymph nodes of the upper respiratory region. P multocida type B:2 is the major cause of HS in Asia, while P multocida type E:2 is the major cause of the same disease in much of Africa. There is a zone in North Africa and the Middle East where both strains are prevalent. According to the OIE reports, it has been reported in the Carribean region and two South American countries. As HS is commonly used as a synonym for shipping fever in these regions, it is unlikely that the HS reported was due to either of the two serotypes. The incubation period is usually two to five days. In peracute infection, death occurs 6 to 48 hours after onset of clinical signs. Vaccines are available. Two doses, 3 to 6 months apart, are given in the first year followed by annual boosters for adequate protection against HS. Probability of semen being infected The bacteria can be found in a range of tissues and there is a presumptive risk of the organism being found in the semen. However, as the bacterium has been recovered from urine samples and other serotypes of P multocida have been isolated from prepuce and semen of healthy dogs50, there is a low risk of semen of carrier donors being infected. Probability of disease transmission via infected semen Transmission of HS is usually by direct contact between animals or through contaminated feedstuff and water. There is no report of HS occurring as a result of artificial insemination with infected 23 semen, nor is there any report on the effects of intrauterine infusion of P multocida. Artificial insemination of livestock is not widely used in HS endemic areas due to the type of farming practices employed. However, other serotypes of P multocida can be transferred by the dog to the bitch at mating with the bitch not being clinically affected by the bacteria transferred.50 Hence it is likely that infected semen can transmit the infective organism to susceptible cows via artificial insemination. Consequences of disease introduction into Australia HS does not occur in Australia. Other strains of P multocida occur in Australia, however serology tests and bacterial isolation tests can differentiate serotypes. HS is regarded as one of the most serious disease of large ruminants in Southeast Asia. Introduction into Australia could have a very significant impact on the livestock industry, especially in tropical northern Australia, where mortalities could be as high as 100%. Because of the occurrence of carrier states, especially in buffalo which is regarded as the most susceptible species, it could prove to be difficult to eradicate. Vaccination would reduce the incidence but add to the costs of production. Estimation of risk There is a very low risk that HS can be transmitted by semen from infected or carrier bulls. Risk management is necessary to prevent the introduction of HS into Australia. Risk management options and recommended measures To minimise the risk of importing HS infected semen, donors need to be clinically and serologically free from P multocida, and not carriers of the disease. As it is difficult to confirm the carrier state, donors must come from areas free from HS. The Code (Article 3.2.12.2.) gives definitions of HS free country and HS free zone. Animals may be introduced into an HS free zone from areas considered infected with HS if certain requirements, including vaccination, are met (Code Article 3.2.12.5.). Vaccination of animals in HS free zones is permitted. As there is no report on the therapeutic effect of vaccination on carriers and as vaccines usually have only preventative value, it is likely that vaccination has no effect on the existing carrier status of the donor. It is proposed the importation of bovine semen be permitted only from countries free from HS (Code Article 3.2.12.2.). BOVINE HERPESVIRUS-1 Bovine herpesvirus-1 (BHV-1) causes several clinical syndromes in cattle including infectious bovine rhinotracheitis and infectious pustular vulvovaginitis. BHV-1 can be subtyped and strains of BHV1.1 and BHV1.2a have been reported to be abortigenic. Incubation period is usually 2 to 4 days but it can be as long as 10 to 14 days. Nasal viral shedding can be detected for 10-14 days after infection. Recovered cattle can continue to shed the virus intermittently. These latent carrier animals may introduce infections into susceptible herds. Animals seronegative to the serum neutralisation test (SNT) can be latent carriers.33 The virus has a worldwide distribution except Denmark and Switzerland where BHV-1 has been eradicated. Eradication programs are underway in some member states of the European Union. BHV-1 is enzootic in Australia. Australian isolates of BHV-1, belonging to subtypes 1.2a and 1.2b, are not known to cause abortions whereas, in North America and Europe, abortion is a common 24 sequela to infections with the respiratory form of BHV-1. It appears that virulent forms of BHV-1 infections are very much the exception in Australia but quite common in North America and Europe. BHV subtype 1.1, usually regarded as being more abortigenic than 1.2a, has not been reported in Australia. BHV-1 can be identified using virus isolation tests or viral DNA detection methods such as the PCR technique. BHV-1 antibodies can be detected with the VNT or ELISA. Probability of semen being infected BHV-1 is one of the most common viral pathogens found in bovine semen. It can replicate in the mucosa of the preputial region in bulls and contaminate the semen during ejaculation. Probability of disease transmission via infected semen There is a demonstrable risk of transmitting BHV-1 by artificial insemination using frozen semen from infected bulls. The virus can cause reproductive disorders in susceptible female cattle. Consequences of disease introduction into Australia The introduction of abortigenic strains of BHV-1 could cause significant reproductive losses and considerable adverse financial effects to stockowners. Estimation of risk There is a demonstrable high risk of transmitting BHV-1 with semen from infected bulls. Risk management is justified to minimise the probability of introducing exotic strains of BHV-1, particularly BHV1.1 and virulent BHV-1.2a. Risk management options and recommended measures Tests to differentiate between strains of BHV-1 are rarely used as not many laboratories have this capability. Thus risk management options to minimise the introduction of virulent strains of BHV-1 require semen to be free from BHV-1. This can be achieved by requiring that: EITHER donor animals were kept in a country or zone free from BHV-1 OR immediately prior to collection donor animals were kept in herds free from BHV-1 OR semen of donor animals was tested free from BHV-1 with an approved virus isolation test. As latent carriers of BHV-1 can be seronegative to the SNT, serology is an unsuitable risk management option. The Code provides definitions of: country or part of the territory of a country free from IBR (Article 3.2.5.2.) and IBR/IPV free herd (Article 3.2.5.3.). In both Articles, requirements for maintenance of free status are given. Donor animals belonging to either one of these two statuses need no further risk management. AI Centres are eligible to become an IBR/IPV free herd as per Article 3.2.5.3. Other risk management options include testing semen for BHV-1 by either two passages in cell culture or by nucleic acid detection. 25 It is proposed that semen meet the Code conditions as given in Article 3.2.5.7. with an additional option of a nucleic acid test (PCR assay) on following suitable extraction of BHV-1 DNA from the semen. BOVINE PESTIVIRUS Diseases caused by bovine pestivirus, commonly referred to as bovine viral diarrhoea virus (BVDV), include bovine virus diarrhoea (BVD) and mucosal disease(MD). There are two biotypes of pestivirus that are serologically indistinguishable. The non-cytopathic (NCP) biotype can infect foetuses that become persistently infected (PI) and cause them to become immunotolerant throughout postnatal life. MD develops only when superinfection with the cytopathic (CP) biotype occurs in PI animals.6 Often PI animals show no symptoms but maintain the disease in the herd by infecting others. Usually PI animals have no antibodies to BVDV. But if they become superinfected with a heterologous strain of BVDV, they can develop neutralising antibodies against the heterologous strain. Not only are there 2 biotypes of bovine pestivirus, there are also 2 genotypes, 1 (BVDV1) and 2 (BVDV2) infecting cattle. Each genotype can be either cytopathic or non-cytopathic. BVDV2 is more pathogenic and virulent strains can cause severe haemorrhaging with high mortality rates. However, most strains of BVDV1 are not virulent and cause almost unnoticeable disease. BVDV1 has a worldwide distribution and is widespread in Australia, infecting a significant proportion of beef and dairy herds. BVDV2 is usually more pathogenic and is known to exist in North America, Japan and some European countries. It is not known to occur in Australia or New Zealand. Probability of semen being infected Bulls can excrete bovine pestivirus in semen during acute and transient infections and also when persistently infected (PI). Probability of disease transmission via infected semen Semen from transiently infected and PI bulls can transmit pestivirus to females under field conditions and has resulted in reproductive losses in susceptible females and in the birth of PI calves. Consequences of disease introduction into Australia The introduction of BVDV2 could result in significant stock losses with haemorrhagic syndrome. Although difficult to eradicate, the disease can be controlled by vaccination. Vaccines are not currently available in Australia. Epidemics do not usually occur with BVDV. Estimation of risk There is a very high risk of transmitting BVDV with semen from infected bulls. Risk management is justified to reduce the risk of collecting bovine semen infected with bovine pestivirus, especially BVDV2. 26 Risk management options and recommended measures In order to minimise the risk of importing semen infected with BVDV, especially BVDV2, only donor animals not persistently or transiently infected with the virus should be eligible. As no country can currently declare freedom from BVDV and as there is a very high herd prevalence of BVDV worldwide, risk management options must focus on the health status of individual donor animals. The only realistic option is for donor animals to be held in quarantine before semen collection and be subject to a virus isolation test (cell culture with immunoperoxidase test, antigen capture ELISA, or nucleic acid detection test) with negative results. The Code provides no risk management options for BVDV. It is recommended donor animals undergo a period of isolation prior to semen collection. During the isolation period, the donor animals should be subjected to a virus isolation test (cell culture with immunoperoxidase test, antigen capture ELISA, or nucleic acid detection test) with negative results. EPIZOOTIC HAEMORRHAGIC DISEASE VIRUS Epizootic haemorrhagic disease virus (EHDV) is an arbovirus belonging to the orbivirus group of viruses. The virus is regarded as an exotic disease in a number of countries. EHDV is closely related to the bluetongue virus and both have similar mode of transmission, incubation and viraemic periods. The 6 strains of EHD (serotypes 1, 2, 5, 6, 7, 8) that occur in Australia are not known to be pathogenic. However, virulent strains occur in North America (EHDV serotypes 1, 2), mostly in deer, and in Japan (EHDV serotype 2) where Ibaraki disease occurs in cattle. Genetic analysis shows different gene sequences between similar serotypes from different countries. The VP3 gene sequence for EHDV-1 (Australia) is 24% different from the North American EHDV-1.51 Probability of semen being infected Being closely related to BT, it is likely that the virus may sometimes be present in the semen of viraemic bulls. Probability of disease transmission via infected semen Being related to BT, it is probable that EHD viraemia can occur in cows inseminated with infective semen. Consequences of disease introduction into Australia The introduction of pathogenic strains of EHD could have very serious long-term consequences if introduced into the areas where suitable insect vectors are found and deer farms occur. Other countries have different strains of non-pathogenic EHD virus yet do not report clinical EHD. Estimation of risk There is a low risk of introducing new strains of EHD virus in semen from countries considered to be infected with EHD virus. Risk management measures are justifiable to minimise the likely risk of introducing new strains of EHD via infected bovine semen. 27 Risk management options and recommended measures The risk management options for EHDV are similar to those for BTV. Hence it is proposed that: EITHER the donors be kept in BTV free, or seasonally free, countries/zones for at least 60 days before commencement of, and during, semen collection; OR the donors be kept in a Culicoides-proof quarantine station for at least 60 days before commencement of, and during, semen collection; OR serum samples be collected from each donor at least 14 days before first semen collection, between 28 days and 60 days after final semen collection, and during semen collection period, at intervals of at least 28 days apart, if there is more than 60 days between the pre-collection and post-collection serum samples; and tested for EHDV antibodies for each serotype of EHD known to occur in that country, with negative results in each case, OR blood samples were collected from each donor at the commencement of semen collection; at the conclusion of semen collection; and either at least every 7 days during semen collection (for a virus isolation test) or every 28 days during semen collection (for a PCR). and subjected to a virus isolation test or nucleic acid detection test (polymerase chain reaction technology [PCR]) for EHDV, with negative results. 28 4. ARGENTINA 4.1 Argentina’s Veterinary Services (as extracted from a Report “Analysis of BSE risk factors in Argentina” prepared by: Dr. B. Cané, Chief Veterinarian, SENASA, Dr. E. Gimeno, FCV-UNLP, Former president OIE (1985.1991). Dr. J.C. Manetti, SELSA-SENASA; Dres C. Van Gelderen and E.Ulloa, SERONO S.A. Argentina and Dr. A. Schudel, INTA-CICV.) “SENASA (Servicio Nacional de Sanidad y Calidad Agroalimentaria), the National Services of Animal Health, is a branch of the Secretary of Agriculture. Its activities are based on the Law of Sanitary Police Nº 3959 for the control of livestock diseases and on the Decree Nº 4238 on Meat and By-products Inspection. SENASA defines the sanitary policy and coordinates its implementation through three Divisions, namely, the Field Service (SELSA), the Slaughterhouse Inspection Service (IPA), and the Laboratory (DICOM). INTA is also a branch of the Secretary of Agriculture. It is administered autonomously by a bureau consisting of livestock farmers, and academic and scientific representatives. Its staff is composed of 4,800 agents distributed over the country, among which 157 are well trained professionals on Animal Health and development related to the agroindustries but they also support SENASA´s programs for animal health. An active interchange with farmers and private professionals is also promoted. Each of the 23 provinces has an animal health administration which organizes the program of activities proposed by SENASA, mainly those concerned with the field control of disease and with the register of veterinarians responsible for health inspection at the municipal slaughterhouses (Federal Meat Law). In turn, each city is responsible for the sanitary control in its administrative district according to the law of Sanitary Police and Decree on Federal Meat Inspection. Argentina’s Animal Health Network Working within a comprehensive legal framework, SELSA (SENASA´s Field Service), has 333 professionals and 1047 technical assistants who control all the farms in the country (more the 300,000) through periodic inspections, and in response to requests for professional assistance; for example veterinary advice was given to 20,832 farmers during November, 1990. The Foot and Mouth Disease vaccination program allows inspection of almost the whole bovine and sheep population of the country two or three times a year. The program was started in 1945 and major improvements were made in 1960 and, again, in 1986. The objective of the program is the eradication of Foot and Mouth Disease and it has the active participation of individual farmers and farmer´s associations. The disease has been eradicated south of the 42nd parallel. However, it is carried out under veterinary supervision. The eradication of Foot and Mouth Disease and the control of sheep scab greatly facilitate the surveillance of the cattle and sheep populations for any emerging disease problems. The movement of all animals (from farms, auctions and to slaughterhouses) is under the control of SELSA´s regional officers, who provide the specific authorizations for such movements.” 29 Control programmes for the following cattle disease are managed by SELSA: “- bovine and sheep scabies (Decree 7383, 1941) - foot and mouth disease (Law 12979, -Decree 5153, 1945) - echinococcosis (Decree 92705, 1941) - bovine paratuberculosis (Decree 5561, 1969) - bovine hipodermosis (Decree 7923, 1964) - bovine viral diarrhea and bovine rhinotracheitis (Decree 406, 1984) - bovine brucellosis (Resolution 698/80, 73/82, 347/86) - bovine tuberculosis (Decree 406, 1984, 347/86, 695/87) - ticks ( Law 12566, Decree 7623, 1954) - rabies (Decree 6134, 1983) IPA, SENASA´s Slaughterhouse Inspection Service, checks the origin and movement of cattle destined for slaughter and performs the ante and post mortem inspections. In addition, they control meat hygiene, including rendered products and the environmental conditions of transport and trade in meat and by-products in relation to human health. IPA has a staff of 1,381 veterinarians, chemists and technical assistants who are assigned to those abattoirs and meat processing plants which are subject to Federal Inspection and whose products are mainly for export. IPA also supervises the veterinary inspection at the local abattoirs which slaughter animals for the domestic market. DICOM, SENASA´s Laboratory Service, is dedicated to the control of veterinary products and the diagnosis of animal diseases. It also acts as a reference laboratory for food processing plants and controls the quality of the analyses performed at its own laboratories. It has a Central and 11 Regional Laboratories with 71 staff professionals to support the disease control programs. DICOM, together with laboratories of INTA, the Provinces and Universities form the Laboratory Network of Animal Health in Argentina.” Some of these laboratories have been designated by the OIE as centres of expertise in leptospirosis, paratuberculosis and bovine tuberculosis. Control of exotic diseases in Argentina The importation of live animals, semen and embryos is strictly controlled by the by the Quarantine and Prevention Department of SENASA. “INTA provides training and services in the diagnosis of exotic diseases to support SENASA activities. Special training courses with international experts are sponsored by the USDA- IICAINTA once a year. These courses include attention to scrapie and BSE. There is expertise at INTA in the clinical and pathological diagnosis of a number of exotic diseases and also in the ultrastructural and biochemical changes associated with these diseases. INTA veterinarians have received training at the following centres of expertise: - Moredun Research Institute, Edinburgh, U.K. - Centre for Tropical Veterinary Medicine, Edinburg, U.K. - Veterinary School, Uppsala, Sweden. - NIH, Bethesda, U.S.A.” SENASA implemented an extremely effective eradication program for FMD which involved developing an effective veterinary services infrastructure, including animal health laboratories, and the establishment of a National Epidemiological Surveillance System. The surveillance system required participation of all sectors of the farming community and the delineation of an effective zoning structure between the vaccinated and unvaccinated zones. All these operations are supported 30 by legislation. Attachment 1 has further detail under “Risk Analysis of FMD in the Argentine Republic.” Further information on Argentina, especially livestock population, the livestock industry, and the animal health infrastructure can be found in the article on Risk Analysis of Foot-and-Mouth Disease (FMD) in the Argentine Republic at Attachment 1. 4.2. Occurrence of disease agents in Argentina Foot and mouth disease virus The last report of FMD in Argentina was in 1994. The OIE 1994 Animal Health Yearbook reported that “the epidemic situation observed in the province of Rio Negro by the end of 1993 was extinguished by March by the use of eradication measures and animal movement control. In 1994 a total of 18 foci was recorded, representing a reduction of 92% when compared to 1993. Same tendency was observed with respect to the number of affected geographical quadrants that decreased in 86%. This is the lowest occurrence of FMD in the history of Argentina’s national program. It is noteworthy that only 4 provinces were affected and that the country registered its last outbreak in April.” Argentina once had a history of a high incidence of FMD as shown in Table 5. Table 5 Year 1985 1990 1992 1993 1994 1995 - 8 No. of FMD outbreaks 1240 841 350 196 18 0 Argentina now meets the Code requirements for a country officially free from foot and mouth disease where vaccination is practised in accordance with Article 2.1.1.2.. It is aiming to become recognised as an FMD free country where vaccination is not practised. Plans are underway to cease all FMD vaccinations from 30th April 1999. Argentina has an ongoing National Program of Eradication to prevent the entrance or spread of FMD. The latest program involves: 1. maintenance of a high level of immunity in cattle north of the Barrancas and Colorado Rivers with a vaccination program (vaccination zone); 2. a monitoring program based on investigation of suspect cases using sero-epidemiological analysis and continuing analysis of the internal and external risk factors; 3. strict control of stock movements; 4. control over imports of animal and animal product. Only cattle have been vaccinated since 1997. Other susceptible stock such as sheep, goats and pigs were not allowed to be vaccinated. 31 SENASA has established the area south of the Barrancas and Colorado Rivers (known as the Patagonia region) as free from foot and mouth disease without vaccination. Argentina has not requested that this area be officially recognised as an FMD free zone where vaccination is not practised in accordance with the Code (Article 2.1.1.2). Detailed information on the foot and mouth disease program in Argentina can be found in the article on Risk Analysis of Foot-and-Mouth Disease (FMD) in the Argentine Republic at Attachment 1. Good quality oil based inactivated FMD vaccines are used in Argentina. There is some concern that vaccination is not effective in FMD carriers. However, it has been over 4 years since the last outbreak and, as carrier cattle only remain persistently infected for up to 42 months, there should no longer be any carriers remaining in vaccinated or unvaccinated cattle in Argentina. Semen from bulls born and reared in - the FMD free zone not practising vaccination in the Patagonia region in the southern portion of Argentina, currently offers the lowest risk. As long as Argentina continue to remain free from FMD and as long as the Code conditions are met, imported semen should present a negligible risk of introducing FMD into Australia. Vesicular stomatitis virus VS is notifiable in Argentina and was last reported in 1986. According to OIE Annual Reports there have been no reports of VS in any countries bordering Argentina except Brazil. Before 1986, the last isolation of the virus was in 1963. However during 1979-80, there was a survey involving 282 serum samples collected from unvaccinated horses over 10 years old. The neutralization test found 33% of these samples positive to the Indiana subtype of VS and 19.3% of samples positive to the new Jersey subtype but the CFT found only 0.35% and 4.4% of these samples positive respectively.9 The sporadic nature of reports of VS in Argentina and the serologic evidence of possible sub-clinical infections is a cause for concern. VS is endemic in countries further to the north, such as Colombia, Venezuela, and Brazil. Bluetongue virus Bluetongue virus (BTV) occurs in many countries lying between 400 N and 350 S. As the northern border of Argentina lies 220 S, some of Argentina lies within the so-called BT zone. Bluetongue has not yet been detected in Argentina. BT is a notifiable disease. Culicoides species are present in northern areas of Argentina. There are suspicions that BT exists in Argentina however no serological evidence or clinical cases of BT have been reported. Other arboviruses including the equine encephalomyelitides (Venezuelan EE, Western EE, Eastern EE, and Saint Louis EE) have occurred in Argentina. Serum samples collected from llamas in Buenos Aires, Cordoba and Jujuy provinces in 1993 were all negative for BT antibodies.26 306 serum samples from cattle herds in the Llanos de La Rioja region were also negative for BT antibodies as were 2490 serum samples from cattle on 70 farms in the Corrientes province.5,20 In 1982, a survey showed BT antibodies in serum of 19.6% of 1752 cattle and from 64 of 99 farms in the Los Lagos area of Chile (39.500S).23 This area is not far from the Patagonia district of Argentina. The occurrence of BT in this part of South America strongly suggests that BT may also occur at least episodically in Argentina. Leptospira spp 32 Leptospirosis is endemic in Argentina. There are over 230 serovars of the pathogenic leptospires recorded worldwide. Routine testing does not allow sufficient differentiation to identify exotic strains of leptospires. Rabies virus Rabies is enzootic in South America. The portion of Argentina north of 290S, home to about 4 million cattle, is enzootic to paralytic rabies transmitted by vampire bats. Peak activity occurs during winter every four years.16,21 Rabies spreads rapidly among vampire bats, causing high mortalities (over 50%) over a short time period.8 Bat populations recover slowly due to the low reproduction rate. Paralytic rabies can occur throughout the year as vampire bats do not usually hibernate or migrate. Vampire bats extended their distribution southwards at 40 km per year during the 1970’s.13 The problem created by these bats depends on the environment. In areas of high livestock density the bats are synanthropic and their populations high. In these areas the bats feed almost exclusively on livestock and paralytic rabies is a serious economic problem. The frequency of outbreaks and the ability of the disease to spread (41 separate outbreaks occurred in addition to an epidemic between 1984 and 1993) have caused heavy losses. In one outbreak reported during 1987-88, 250 cattle and 60 horses died. Over the 10 year period, about 30 000 cattle died from paralytic rabies. In areas of low livestock density, the vampire bat population is less dense. They feed on various species of animals but paralytic rabies occurs only sporadically with one isolated outbreak between 1984 and 1993.8 Paralytic rabies from vampires, commonly referred to as the bat variant, occurs in the northern part of Argentina (15% of the country). Patagonia appears to be free from rabies.15 Consumption of dead and dying bats by carnivores, particularly foxes and skunks, is suspected to cause further spread. The dog variant of rabies also exists in Argentina. In 1979, the province of Buenos Aires recorded a total of 714 cases of animal rabies, 86% in dogs, 12% in cats and 2% in other species. This represents a fall of 85% from the number reported in 1976.1 This variant is not considered to be important in cattle. Argentina has programs to eliminate the risks to public health and to reduce cattle losses in risk areas. There is a preventative vaccination program in risk areas and programs exist for monitoring and controlling the vampire bat population. Mycobacterium paratuberculosis Although this disease has been reported in Argentina since the early 1970’s, its distribution is not known and there are no organised regional control programs. Brucellosis abortus Brucellosis (Br) is enzootic in Argentina. Argentina has embarked on a national program to eradicate Br. This program involves 1. Br being declared a notifiable disease; 2. vaccination of calves between 3 and 10 months; 3. control of cattle movements; 4. eradication of the disease from establishments using accredited veterinarians; and 5. implementation of agreements for eradication in dairy herds. The number of vaccinated calves rose from 2,890,000 in 1992 to 4,590,000 in 1997 (out of a total of approximately 55 million cattle). Apparently the program calls for vaccination of all calves. Br has 33 been eradicated from number of farms and in 1997, 2 427 farms in the Santa Fe Province were certified to be free from Brucellosis Mycobacterium bovis Tb, a notifiable disease, is enzootic in Argentina. A national control program based on the voluntary eradication of the disease is in place. Eradication strategies are being redefined, depending on the prevalence of Tb in different areas. The program however basically involves the intradermal tuberculin test for Tb every 60-90 days, and the slaughter of reactors. Once a herd has undergone two consecutive negative tests, only annual testing is required. According to SENASA’s 1997 Annual Report, of 9,472,396 animals (representing 47.36% of the animals tested from 3/95 to 2/97), 128,038 (1.3%) were positive and had Tb-like lesions. Bovine leukemia virus . In Argentina, EBL occurs in both beef and dairy herds. Prevalence is very high in some areas such as in dairy herds around Buenos Aires but much lower in areas such as Patagonia where 3 000 bovine serum samples collected from a number of farms were all negative.41 Pasteurella multocida (Serotypes B:2 and E:2) HS has not been reported in Argentina and is a notifiable disease in that country. Bovine herpesvirus-1 BHV-1 is enzootic in Argentina. The disease occurs throughout the country with a prevalence of 36% to 66%. BHV-1 viruses have been isolated from aborted bovine foetuses. A study of the viral isolates from genital infections showed BHV-1.1 and BHV-1.2 characteristics.25 Antigenic patterns of BHV-1 isolates from genital infections from Argentina, examined with monoclonal antibodies generated with BHV-1.1 strains, showed BHV-1.1/2 characteristcs.22 Bovine pestivirus BVDV2 has not been reported in Argentina. However, BVDV2 has recently been detected in southern Brazil4 and it is likely that it occurs in Argentina as well. Outbreaks of bovine viral diarrhoea with high mortalities have occurred. The epidemiology of these outbreaks suggests that BVDV2 was the cause. One such outbreak occurred on a farm in 1991 in the Santa Fe province and lasted 3 to 4 months. A summary of this outbreak is given in Table 6. Description steers 7 - 12 months heifers 1- 2 years heifers 2 - 3 years Table 6 Total No. Deaths 300 14 1370 53 1530 4 Total affected (incl. deaths) 80 150 20 Epizootic haemorrhagic disease virus 34 As EHD and BT viruses are closely related with EHD being found in the same ecological conditions as BT, refer to section 4.1.7 for possible occurrence of BT in Argentina. In the same way, it is suspected that EHD may occur in Argentina. 35 4.3 Summary of risk management measures Table 7 DISEASE OIE List Level of risk management necessary Type of management necessary Foot and mouth disease A 010 Negligible Certification of disease freedom and test as necessary Vesicular stomatitis A 020 Low Certification of disease freedom Rinderpest A 040 Nil Certification of disease freedom Contagious bovine pleuropneumonia A 060 Nil Certification of disease freedom Lumpy skin disease A 070 Nil Certification of disease freedom Rift Valley fever A 080 Nil Certification of disease freedom Bluetongue A 090 Low Tests Leptospirosis B 056 High Certification of antibiotic inclusion Rabies B 058 Very low Certification of absence of disease Johne’s disease B 059 High Tests Bovine brucellosis B 103 High Tests Bovine tuberculosis B 105 High Tests Enzootic bovine leucosis B 108 High Tests Haemorrhagic septicaemia B 109 Nil Infectious bovine rhinotracheitis B 110 Moderate Tests C 652 High Tests - Negligible Tests OIE List A diseases OIE List B diseases Certification of disease freedom Other diseases Bovine virus diarrhoea Epizootic haemorrhagic disease 36 5. 5.1 BRAZIL Brazil’s Veterinary Services The Conselho Federal de Medicina Veterinária, reports there being 56,214 veterinarians and 41,015 animal technicians registered in Brazil in 1997. The organisation which is responsible for Brazil’s veterinary services is the Departmento de Defesa Animal in the Ministerio da Agricultura do Abastecimento e da Reforma Agraria (MAARA). The department carries out government policies for the provision of animal health services through monitoring and surveillance of livestock diseases; provision of front line veterinary services; supervision of international and interstate movement of animals, animal products, and veterinary pharmaceutical and biological products; supervision of the production, distribution and use of veterinary pharmaceutical and biological products; promotion of educational programs on livestock health; provision of veterinary laboratory services and inspection of animals and animal products. Some states, eg, State of Minas Gerais, have passed legislation requiring the obligatory vaccination of livestock against foot and mouth disease, bovine brucellosis and rabies. It appears that the veterinary services in the southern states are much more organised than in the less developed north. Certainly the FMD program is further advanced in the southern states with the two southernmost states being declared free from FMD with vaccination in 1997. Also, prompt response to the FMD outbreaks and the obligatory vaccination of cattle in the past few years in the central west states have resulted in a rapid decline of FMD in these areas and attest to the efforts of MAARA to eradicating FMD. 5.2. Occurrence of disease agents in Brazil Foot and mouth disease virus FMDV occurs in Brazil. The far south states of Santa Catarina and Rio Grande du Sol are recognised as free from FMD where vaccination is practised. Elsewhere, the incidence of FMD has dropped dramatically in recent years, as shown below in Table 8. Year 1993 1994 1995 1996 1997 Table 8 No. of outbreaks 1417 2084 666 200 167 Two outbreaks from the Mato Grosso State occurred in February 1998 near the Paraguay border. Just recently (January 1999), an outbreak was reported in the same state, but in the far southern area. 37 A massive FMD eradication program is underway with extensive monitoring and surveillance systems being established. It is planned to eradicate FMD from Brazil by the year 2009. Vesicular stomatitis disease virus The Indiana strain of VSV occurs sporadically in Brazil. It was last reported in 1996 when thirteen outbreaks were reported. Twelve occurred in the state of Minas Gerais and one in Mato Grosso du Sol. Bluetongue virus BTV is endemic in Brazil. There is very little information on BT serotypes found in South America. However, antibodies to BT serotypes 4 and 20 were detected in cattle from Brazil being held in quarantine in Florida. BT serotypes 6, 12, 14 and 17 have been reported in neighbouring Colombia and Surinam. No reports of clinical bluetongue could be found in South America. Leptospira spp Leptospirosis is endemic in Brazil. Serosurveys suggest that most serogroups occur in Brazil, being found in snakes, wild forest animals, humans and domestic animals.42 Leptospiras isolated from bovine foetuses include L. interrogans svs hardjo, pomona and wolffi.43 Other isolates recovered from infected cattle include L. santarosai svs goiano and guaicurus.44 80% of semen samples from 20 bulls at AI Centres in Brazil were positive for leptospirosis by the PCR.45 Rabies virus Several variants of rabies, most notably the dog variant and vampire, insectivorous and frugivorous bat variants, are endemic in Brazil. Heavy livestock losses due to outbreaks of vampire rabies have been reported. Annual vaccination of cattle against rabies, especially against vampire bat rabies, has significantly reduced stock losses in recent years. Mycobacterium paratuberculosis Johne’s disease has not been reported in Brazil. It is not known whether any surveys for Johne’s disease have been conducted. However, the presence of Tb in cattle can present problems in diagnosing JD. Brucella abortus Bovine brucellosis is endemic in Brazil. There appears to be no official eradication programs in place. Some of the larger stockowners have controlled Br in their herds at their own expense. While vaccination with Strain 19 is obligatory in some states, there are large areas of Brazil, especially in the Amazonian region, where Br prevalence is very high. There is very limited surveillance or control activity in these areas. Mycobacterium bovis Tb is endemic in Brazil. Brazil does not appear to have carried out either large scale tuberculin testing or national sampling surveys to determine the prevalence of Tb. The prevalence of Tb is believed to be over 1%. Tb tuberculin tests performed during 1986 in four regions of the country 38 showed variations in the level of infection ranging from 0.9 to 2.9% while 6.2 to 26.3% of the tested herds harboured reactor animals. Condemnations due to Tb like lesions at slaughterhouses seem to be remarkably lower in Brazil than in Argentina.7 Using DNA restriction fragment length polymorphism, Tb isolates from Brazil presented patterns different from isolates from Argentina, Paraguay, Mexico and Netherlands.12 Bovine leukemia virus EBL occurs in cattle in Brazil. Sero-epidemiological surveys for EBL have been conducted in the States of Rio de Janeiro46 where over 54 % of dairy cows surveyed had antibodies to EBL and in Minas Gerais47 where 23% of embryo donor and recipient cows surveyed had antibodies to EBL. Pasteurella multocida (Serotypes B:2 and E:2) According to the OIE Annual Report, HS occurs sporadically in Brazil and Venezuela. There are no published reports of HS due to P multocida serotypes B:2 and E:2 in livestock in South America and it is doubtful whether true HS does occur in South America. HS is commonly used as a synonym for shipping fever in Central and South American countries. Bovine herpesvirus-1 BHV-1 is enzootic in Brazil. As pathogenic BHV-1.1 and BHV-1.2 occurs in Argentina, it is presumed that these strains occur in Brazil also. Bovine pestivirus The prevalence of BVDV infection in Brazil appears to be similar to that found in Europe and the USA. BVDV2 has been reported in southern Brazil.4 Epizootic haemorrhagic disease virus Cattle with neutralising antibodies to EHD serotypes 1 and 2 have reported in northern Colombia. It is most likely that EHDV can be found in Brazil. 39 5.3 Summary of risk management measures Table 9 DISEASE OIE List Level of risk management necessary Type of management necessary Foot and mouth disease A 010 Very low Certification of disease freedom and test Vesicular stomatitis A 020 Low Certification of absence of disease Rinderpest A 040 Nil Certification of disease freedom Contagious bovine pleuropneumonia A 060 Nil Certification of disease freedom Lumpy skin disease A 070 Nil Certification of disease freedom Rift Valley fever A 080 Nil Certification of disease freedom Bluetongue A 090 Low Tests Leptospirosis B 056 High Certification of antibiotic inclusion Rabies B 058 Very low Certification of absence of disease Johne’s disease B 059 High Tests Bovine brucellosis B 103 High Tests Bovine tuberculosis B 105 High Tests Enzootic bovine leucosis B 108 High Tests Haemorrhagic septicaemia B 109 Negligible Certification of disease freedom Infectious bovine rhinotracheitis B 110 Moderate Tests C 652 High Tests - Negligible Tests OIE List A diseases OIE List B diseases Other diseases Bovine virus diarrhoea Epizootic haemorrhagic disease 40 6. 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J Am Vet Med Ass 179: 2, 169-171. 41 Huici N, Segade G, et al (1995) Seroprevalence of enzootic bovine leucosis in Patagonia Veterinaria Argentina 12: 115, 303-305 42 Lins ZC; Lopes ML (1984) Isolation of Leptospira from wild forest animals in Amazonian Brazil. Trans R Soc Trop Med Hyg 78: 1, 124-126 43 Langoni H; de Souza LC; da Silva AV; Luvizotto MC; Paes AC; Lucheis SB (1999) Incidence of leptospiral abortion in Brazilian dairy cattle. Prev Vet Med 40: 3-4, 271-275. Santa Rosa CA; Sulzer CR; de Castro AF; Yanaguita RM; Giorgi W (1980) Two new leptospiral serovars in the Hebdomadis group isolated from cattle in Brazil. Int J Zoonoses 7: 2, 158-163. 44 45 Heinemann MB; Garcia JF; Nunes CM; Morais ZM; Gregori F; Cortez A; Vasconcellos SA; Visintin JA; Richtzenhain LJ (1999) Detection of leptospires in bovine semen by polymerase chain reaction. Aust Vet J 77: 1, 32-34. 46 Romero CH; Rowe CA (1981) Enzootic bovine leukosis virus in Brazil. Trop Anim Health Prod 13: 2, 107-111. 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Am J Vet Res 53: 5, 670-673. 51 Weir RP, Harmsen MB, Hunt, NT et al (1997) EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in Northern Territory. Vet Microb 58: 135-143. 44 Attachment 1. RISK ANALYSIS OF FOOT-AND-MOUTH DISEASE (FMD) IN THE ARGENTINE REPUBLIC CONTENTS Introduction Livestock Population The Livestock Industry Health Infrastructure National FMD Eradication Plan Epidemiological SurveillanceEmergency System Residual Viral Activity Biosafety External Risk Laws and Regulations Flow Chart INTRODUCTION On May 30th , 1997, the International Committee of the Office International des Epizooties, in Paris, unanimously approved the recognition of Argentina as "Foot and Mouth Disease free country where vaccination is practiced". This sanitary status was achieved after implementing a successful strategy to control and eradicate the disease on and after 1990. However, the acquired condition is temporary since we aim to be recognized as a "Foot and Mouth Disease free country where vaccination is not practiced", condition of great impact for the livestock industry and also for the whole country. From the animal health policy and international trade viewpoint, it represents a transcendental qualitative change, since it implies that a great beef producer country could enter the international market of countries free of "Foot and Mouth Disease". From the technical aspect, this change in the qualitative status requires to count in the short term with strategies and technical, human and economic resources to decrease the risk of the disease reappearance and to detect, control and eliminate the disease in case of an eventual occurrence. Risk Analysis (including risk identification, evaluation, management and communication) is a scientific methodology already applied within the scope of economics and engineering, but it has been recently used in veterinary medicine. The identification of key events composing each of the risks allows taking preventive measures to decrease the likelihood of risk occurrence and therefore, control and eradicate the disease. The purpose of this report is to qualitatively assess the risk of a Foot and Mouth Disease case ocurrence. Analyzing completely and exhaustively the present situation to determine which are the predominant risk factors. Recognizing risk factors will allow the adoption of effective and efficient measures to prevent, control and eliminate the disease in an eventual case. 45 The main factors participating in the risk of Foot and Mouth Disease occurrence have been evaluated in this qualitative analysis. Those factors are: the characteristics of the susceptible livestock populations, slaughter and processing industry, dairy industry, animal health infrastructure, organization of the National Foot and Mouth Disease Eradication Plan 1993-1997 and the epidemiological surveillance systems, including the emergency system. The viral activity in the field and the biosafety in the laboratories dedicated to research and vaccine production against Foot and Mouth Disease have been analyzed as internal risk factors. As external risk factors, it has been considered the Foot and Mouth Disease situation in the five neighbouring countries, animal and by-product imports from neighbouring and non-neighbouring countries, passengers, planes and ships movements in border points, airports and ports, and the infrastructure and available resources for internal risk reduction. Finally, legislation (laws, decrees and resolutions) related to Foot and Mouth Disease has been considered: regulations of the national eradication plan, animal movement, and slaughterhouse control and animal and by-products imports. The conclusion of this analysis permits to qualitatively identify the main risk factors, mistakes and needs of preventive actions to be implemented. As a second step of this investigation, the magnitude of these risks has to be determined. LIVESTOCK POPULATION The livestock population on commercial operations in Argentina totals 80 million heads of domesticated animals susceptible to Foot and Mouth Disease (FMD) (cattle, sheep, swine and goats), in addition to 780,000 South American camelidae and 14,000 deer. The cattle population is mainly located in the humid pampas and the north-east of Argentina. There are 270,000 operations which on average have 208 heads, 54.4% of the bovine population is owned by 11,3% of the farmers who have over 500 heads. There are different types of production areas, each with its particular agroecological condition: a fattening area in the West of the Province of Buenos Aires, South of Santa Fe and south-east of Córdoba; and two breeding areas: the Salado River Basin (to the East of the Province of Buenos Aires) and the north-eastern part of Argentina. The number of heads of cattle as well as the beef production has remained stable in recent years (5055 million heads and approximately 2.5 million tones). Eighty per cent of the production is for domestic consumption and the remaining 20% is exported. There are approximately 20,000 dairy farms which together have a population of 2 million cows. These operations are mainly located in the areas of Córdoba-Santa Fe, Abasto in Buenos Aires, the maritime Atlantic coast and hills of the Province of Buenos Aires, Trancas-Tucumán, Paraná, and the eastern part of the Province of La Pampa. In recent years, dairy production has experienced a sustained growth. Pig farms are mainly found in the corn belt area (north of Buenos Aires, south of Santa Fe and Córdoba), and in the dairy production area of Córdoba-Santa Fe where the milk processing plants are located, these operations use large amounts of milk by-products (whey) as pig feed. Sheep production is declining. There are approximately 30,000 farms which on average have 655 heads. These operations are mainly concentrated in three regions: Patagonia, Buenos Aires, and the Mesopotamia. 46 Goat farms are found in marginal areas, from the agroecological point of view, with subsistence farming. They are located by the foothills of the Andean range, in the semi-arid valleys of the northern part of the country, and the semi-arid central region of Argentina (hills and the Chaco area). Camelidae are mainly on the semi-arid plateau of Patagonia, the high plateau of the northwest (Jujuy, Salta, Catamarca), and in some semi-arid valleys in the North of Argentina. Bovines are not usually found with other susceptible species, except in some areas of the Province of Buenos Aires and the Mesopotamia, where farmers have both sheep and cattle. It is unusual to find susceptible feral species with cattle. Only the four domesticated species were involved in the FMD outbreaks in Argentina, and they were mainly bovines. Feral species have never been affected by any FMD outbreak. The recent opening up of the Argentine economy led to a more intensive beef cattle production, although this occurred much earlier in the dairy sector. THE LIVESTOCK INDUSTRY SENASA has licensed 183 meat-packing plants to slaughter cattle, 30 for sheep, and 64 for pigs. In total, SENASA’s staff includes 669 veterinarians and technicians in those plants. In 1996, these plants slaughtered 10.9 million cattle, 571 thousand sheep and 1.6 million pigs. In addition there are over 200 plants which have been licensed by the Provinces or the Municipalities to slaughter cattle and other species. The meat-packing plants which are licensed either and controlled by SENASA slaughter eighty-five per cent of the cattle slaughtered in the country, and meet the requirements for processing pathological and non-pathological residues (solid and liquid). In addition, ante and post mortem controls are mandatory to identify any clinical signs or lesions caused by FMD. Argentina has 890 milk processing plants, which have a daily installed capacity of 23 million litres. Regulations require that only pasteurized milk be used for soft and semi-hard cheese manufacture; this requirement does not apply for hard cheeses. The pH of the whey in the cheese reaches between 5.2 and 6, which does not limit bacteria development or inactivate other micro-organisms. Most of this whey is used as pig feed. In 1996, 2 million litres of whey were used as pig feed. Fifty-seven per cent of this whey was from non-pasteurized milk, and this could potentially be a health hazard. There were no FMD outbreaks caused by whey consumption, therefore given the current epidemiological situation, it would be true to say that the is negligible probability of whey being a source of FMD virus for pigs. HEALTH INFRASTRUCTURE SENASA is undergoing an institutional transformation as a result of the creation of the new Agency which was formed by merging the former SENASA (Animal Health Service), the IASCAV (Plant Health Service) and the agrifood sector. SENASA is a decentralized Agency that reports to the National Secretariat of Agriculture, Livestock, Fisheries and Food. It is a Federal Agency and its rules and regulations are enforced throughout the country. It has 2,993 employees who are responsible for issuing rules, inspecting, certifying, implementing and recording Animal Diseases throughout the country. Its actions are enforced through six National Bureaus, five Regional Bureaus, and other National, Regional, Provincial, Municipal and International bodies as a result of agreements, pacts and/or branch offices. The National Animal Health Bureau, the Bureau of Laboratories (DILAB) and 47 the National Bureau for Agrifood Inspection are mainly responsible for implementing SENASA’s effort to eradicate FMD. It also has the support of other National Bureaus and SENASA’s five Regional Bureaus. The institutional readjustment necessarily requires an in-depth analysis of SENASA’s structure and functions, to make it more efficient. NATIONAL FMD ERADICATION PLAN SENASA is the National Agency responsible for Controlling and Eradicating FMD. The implementation of the 1990-92 FMD Control Plan and the 1993-1997 Eradication Plan have enabled Argentina to achieve a status that has been recognized by the International Committee of the Office International des Epizooties (OIE) as "FMD Free with Vaccination" as of May 30, 1997. This achievement was the result of the successful actions that were developed by the FMD control and eradication plans, the active participation of the farmers, and the use of an high quality vaccine. Law 24,305 states that SENASA is the Agency responsible for defining the national eradication strategy, and grants authority to the National FMD Eradication Committee to plan, follow-up and assess the FMD eradication program. The operational strength of the control and eradication plans is mainly based on the participation of the Provincial organizations (COPROSA’s), 350 Local Animal Health Committees, and the epidemiological surveillance and health enforcement actions developed by SENASA. There are other National (INTA, CEVAN), Provincial and Municipal agencies that actively participate in the National Plan and that have the cooperation of private veterinarians and others who are responsible for surveillance and control. At present, Argentina has two regions which have a different health status: the area to the North of the Negro River and the Province of Neuquén which are FMD Free with vaccination, and the one to the South of this area which is FMD free without vaccination. All shipments between these two areas must comply with the requirements established by SENASA (Res. Nº 506/97). The number of vaccinated bovines increased significantly until 1993, and then stabilized. This was key for the success of the Plan. Before 1985, the number of vaccinated bovines did not exceed 45 million heads; at present, over 55 million are vaccinated. Some marginal areas have special plans, and the vaccine coverage might be improved. As of 1994, vaccination of sheep has not been mandatory, and since May 1997, no sheep, pigs or other susceptible species are allowed to be vaccinated (Res. Nº 88/97). The excellent vaccine coverage gave high immunity to the livestock population in Argentina. This was confirmed with the results of the samples taken from two age groups in two Provinces of the central region of the country which were subject to liquid phase ELISA testing. EPIDEMIOLOGICAL SURVEILLANCE To detect any suspect case of FMD at an early stage, the National Epidemiological Surveillance System requires the participation of all the sectors of the farming community. Surveillance includes actions on the agent, the host and the environment. To detect and isolate the agent, the reference laboratory is the DILAB which, together with CICV (INTA), CEVAN 48 (CONICET) and CPFA (OPS), control the characteristics of the field viruses and the vaccine strains. The system continuously monitors the production systems, shipments, concentration markets and marketing, and establishes health-hygiene controls for shipments and slaughter, and the requirements to import animals, semen, semen and other products and by-products. In this new stage and to conclude the Eradication Plan, only continuous education will allow SENASA personnel and the various organizations involved, to achieve higher operational efficiency in the different sectors and a better awareness of the new status and the sensitivity level of the surveillance system. EMERGENCY SYSTEM SENASA has established regulations for zoosanitary emergencies. However, in occasions, there were some difficulties and delays due to juridical troubles. Law 24,305, together with other regulations, seeks to eradicate FMD, and requires an emergency fund to compensate the farmers and cover other operational costs. The current situation indicates that it is indispensable to create and maintain a permanent fund for sanitary emergencies which involve animals, that is additional to the budget. It is important to design a continuous education system for sanitary emergencies for official and private professionals. The system should include training courses, seminars and simulations, as FMD is an exotic disease in Argentina since May 30, 1997. RESIDUAL VIRAL ACTIVITY Viral activity was assessed by analyzing the outbreaks, the results of the seroepidemiological monitoring, the reported and suspect cases of FMD, the inspections at the slaughtering plants and concentration markets, and the identification of animals that could be carriers of the virus. After the implementation of the National 1990-92 Control Plan, viral activity dropped, as shown by the decline in the number of outbreaks in the Provinces in which, historically, the disease was endemic, and the absence of outbreaks in the Provinces in which it appeared sporadically. The declining trend continued during the period in which the National 1993-97 Eradication Plan was in place. As a result of these actions, there were no outbreaks in the Mesopotamia area since 1993 and in the rest of the country since April 1994. The preventive actions which were implemented to maintain the FMD free status in the Southern part of Patagonia and in the Province of Neuquén, were adequate. The appearance of an outbreak in the area of San Carlos de Bariloche in 1993 and 1994, showed that the prevention system was not flawless. However, the absence of outbreaks in the San Carlos de Bariloche region after the emergency actions were put in place confirms that they were efficient to control and eliminate the virus in that region. The results of the serological monitoring performed in the spring of 1996 and the fall of 1997 confirmed the absence of viral activity in the region which is not subject to vaccination. In the Provinces with vaccination, the frequency and distribution of the EITB reactors in cattle between the ages of 6 and 12 months, and of the VIA reactors in other susceptible species (sheep, goats, swine, lamas and deers from commercial herds) do not show viral activity. Samples taken from cattle under one year of age (not vaccinated) in the spring of 1996, confirm the absence of viral activity in the Provinces which were tested. 49 Potency tests performed by the DILAB and the field observations indicated that vaccination may cause antibodies not only for the 3D non-structural protein (VIA) but also for other non-structural proteins. Given the available results, the appropriateness of a massive sampling and the techniques that should be used in the future to detect any viral activity in vaccinated and non-vaccinated animals, should be reviewed. After the last reported outbreak, no diseased animals have been detected by inspections at the slaughtering plants and livestock concentration markets. There were no confirmed cases of infected animals after the reported and suspect cases were investigated; also all the Probang test carried out to detect carriers among the animals that were shipped to the Mesopotamia area and the region which is "Free without vaccination" were negative. The number of notifications of suspected cases of FMD could be considered low, given the number of heads in the livestock population and the pathologies that exist in Argentina and that could be misdiagnosed as FMD. In most cases, differential diagnosis for FMD was based on a clinical examination. From 1997 the number of notifications has been increased significantly, most of the differential diagnosis were based on analysis of samples sent to laboratory. BIOSAFETY The absence of FMD outbreaks requires biosafety measures for diagnostic, research and FMD vaccine manufacturing laboratories: a committee was created to define the biosafety standards, in accordance with internationally accepted criteria. A register was developed for those who store and manipulate the FMD virus; the viruses in possession of the laboratories that did not decide to make the necessary adjustments required by the biosafety standards, were destroyed. The viruses stored at the laboratories which were modifying their facilities as required by the standards to apply for a license, were held in custody. At present there are three commercial laboratories licensed to manufacture the FMD vaccine (BayerSan Jorge-Bagó, Rhone Merieux, and Biogénesis-Syntial) as required by the biosafety standards. One of these laboratories is located in the city of Buenos Aires, and the other two in the suburbs, at a distance of 30 and 50 km from Buenos Aires, in a location where there are no farming operations in the area. There are two official laboratories: INTA (Castelar), and DILAB (SENASA), which are modifying their facilities to apply for a license. In the last five years, 450 million doses of FMD vaccine were manufactured, and 92% of them were approved after passing the official tests. No vaccines were rejected because they were unsafe. The reduction in the number of laboratories that manipulate the FMD virus, the enforcement of biosafety standards for these laboratories, and the absence of rejected vaccines for reasons of unsafety, guarantee the existence of limited stocks of FMD virus and confirm that the internal and external controls for FMD vaccine production, are reliable. EXTERNAL RISK All products or by-products of animal origin imported to Argentina are subject to revision and authorization by SENASA according to international regulations (OIE). 50 Argentina’s border is 14,046 km long, and 9,371 km are shared with five other countries. The rest is Atlantic coastline. The main points of entry into the country are 7 international airports, 26 border crossings, 12 seaports, and 7 international bridges. The port and airport of Buenos Aires concentrate more than ninety percent of all air and maritime imports. In both places, more than 148 Tn of residues are produced daily, they are disposed by burial through only one company. The interdicted biological materials in the airport, are previously denaturalized. The other 11 ports are mainly fishing ports. The remaining international airports operate mainly with flights from the neighbouring countries. SENASA has 114 employees working at the borders They are responsible for controlling the international trade. At the most important points of entry, SENASA has full-time employees who work with Customs and Border Patrol officers. The five neighbouring countries have a total susceptible population to FMD of 293 millions heads. Chile has been FMD free since 1987. Along the 5,000 km of the border with Chile, there are 23 important border crossings, and 158 locally used crossings. Chile has a surveillance system to control the animals that are taken up into the mountains during the summer season. Uruguay has been FMD free since 1995. The last outbreak occurred in June 1990. The 866 km border with Uruguay is all rivers (Uruguay and the Plata Rivers). There are three bridges and seven river ports that receive international trade. Uruguay has a system of barriers to control all entries, particularly from Brazil. Paraguay’s health status is similar to that of Argentina: FMD free with vaccination. Paraguay has had no outbreaks since September 1994. Argentina has a river front of 1,570 km with Paraguay along which there are three bridges and 20 river ports that receive international trade. Two Southern States of Brazil (Río Grande do Sul and Santa Catarina) have had no outbreaks since 1993, and submitted a request to the O.I.E. in May 1997 to be recognized as FMD free with vaccination. Paraná has had no outbreaks since may 1995. Argentina shares 1,079 km with Brazil, of which 27 km are land, and the rest are rivers. There are four bridges, 9 ports and one international crossing. Of the five countries with which Argentina shares a border, Bolivia is the only one where FMD is endemic. Bolivia does not have a FMD control program. In 1996, it had 41 outbreaks. Argentina’s border with Bolivia is 765 km long, of which 380 km are in the high mountains and ravine area, which is difficult to access and control. There are 3 bridges and 4 international crossings. Uruguay, Brazil, Paraguay, Argentina , and recently Bolivia (1997) belong to the Convenio Cuenca del Plata, that started its activities in 1987 with the coordination of CEPAFA. (OPS). This agreement operates under the frame of the Comité Hemisférico de Erradicación de la Fiebre Aftosa (COHEFA) and coordinates at the subregional level, all the efforts for control and eradication of FMD. No susceptible animals or reproductive materials have been imported from Bolivia and Brazil, since 1994. Similarly, no animal products and byproducts were imported from Bolivia, since that date. However, animal byproducts are usually imported from the other four neighbouring countries. The globalization in trade and transit of people and goods constitute the highest risk of reintroduction of FMD to Argentina, there for there is an urgent need to strength all preventive measures to minimize the risk. 51 LAWS AND REGULATIONS The Foot and Mouth Disease Eradication Plan is based on a legal framework that includes many regulations which are the basis for the various actions required by the Plan. Some of these regulations could be considered "Basic" not only because their content is very important, but because they grant authority to SENASA to issue specific rules. The FMD Eradication Plan was developed on the basis of what is stated in Law 3959, Decree 4238/68, Law 21,740/78, Law 22375/81, Law 24,305 and Decree 643/96, which have been continuously updated by resolutions. The system allows adjustments to the operational aspects of SENASA’s work and regulates the issues which are not included in the original laws because of the improvement in the country’s health status. An analysis of the rules and how they apply to specific cases, to assess the operational aspects of the FMD Eradication Plan, indicates that: 1. The legal framework which includes the current Laws, Decrees and Resolutions is appropriate to meet the goals set forth by the FMD Eradication Plan. It must be noted that it is not possible to systematize and include in only a few regulations the various issues required to implement the Plan. 2. The continuous updating of laws and regulations, as a method, allows adjustments as required by the new status. 3. The operational part of the Plan, as described in the regulations, is not always simple because, although the necessary rules are in place, reality offers a wide scope of unforeseen situations. 4. The current regulatory framework optimizes the resources and avoids unnecessary duplication of work. It provides a better integration of the roles and responsibilities of the National Government, the Provinces and the Municipalities. The optimum mechanism to achieve full integration would be one which is based on agreements among those involved. 5. Argentina, with its new health status -FMD free with vaccination-, must amend some specific regulations and in other cases, issue the necessary resolutions. 52 Attachment 2 OIE INTERNATIONAL ANIMAL HEALTH CODE FOOT AND MOUTH DISEASE Article 2.1.1.2. FMD free countries where vaccination is not practised To be listed in "FMD free countries where vaccination is not practised", a country should: 1) have a record of regular and prompt animal disease reporting; 2) send a declaration to the OIE that there has been no outbreak of FMD and no vaccination has been carried out for at least 12 months, with documented evidence that an effective system of surveillance is in operation and that all regulatory measures for the prevention and control of FMD have been implemented; 3) have not imported animals vaccinated against FMD since the cessation of vaccination. The name of the country will be included in the list only after acceptance of submitted evidence by OIE. FMD free countries where vaccination is practised To be listed in "FMD free countries where vaccination is practised", a country should: 1) have a record of regular and prompt animal disease reporting; 2) send a declaration to OIE that there has been no outbreak of FMD for the past two years, with documented evidence that: a) an effective system of disease surveillance is in operation and that all regulatory measures for the prevention and control of FMD have been implemented, and b) routine vaccination is carried out for the purpose of the prevention of FMD and that the vaccine used complies with the OIE standards, and 3) have a system of intensive and frequent surveillance for detection of any viral activity. The name of the country will be included in the list only after acceptance of submitted evidence by the OIE. If an FMD free country where vaccination is practised wishes to change its status to FMD free country where vaccination is not practised, a waiting period of 12 months after vaccination has ceased is required. FMD free zones where vaccination is not practised An FMD free zone where vaccination is not practised can be established in an FMD free country where vaccination is practised or in a country of which parts are still infected. The free zone is separated from the rest of the country and from neighbouring infected countries by a surveillance zone, or physical or geographical barriers and animal health measures which effectively prevent the entry of infection. A country in which an FMD free zone where vaccination is not practised is to be established should: 1) have a record of regular and prompt animal disease reporting; 2) send a declaration to the OIE that it wishes to establish an FMD free zone where vaccination is not practised, where there has been no outbreak of FMD for the past two years, where no vaccination has been carried out for the past 12 months and that no vaccinated animals have been introduced into the zone since the cessation of vaccination; 3) supply documented evidence that an effective system of surveillance is in operation in the FMD free zone where vaccination is not practised as well as the surveillance zone if applicable; 4) describe in detail: a) the boundaries of the FMD free zone, and the surveillance zone, where vaccination is not practised; b) the system for preventing the entry of infection into the FMD free zone; and supply evidence that these are properly supervised and that all regulatory measures for the prevention and control of FMD have been implemented. The free zone will be included in the list of FMD free zones where vaccination is not practised only after acceptance of submitted evidence by the OIE. 53 FMD free zones where vaccination is practised An FMD free zone where vaccination is practised can be established in a country with a free zone where vaccination is not practised or in a country of which parts are still infected. The free zone where vaccination is practised is separated from the rest of the country and, if relevant, from neighbouring infected countries by a buffer zone, or physical or geographical barriers and animal health measures which effectively prevent the entry of infection. A country in which an FMD free zone where vaccination is practised is to be established should: 1) have a record of regular and prompt animal disease reporting; 2) send a declaration to the OIE that it wishes to establish an FMD free zone where vaccination is practised, where there has been no outbreak of FMD for the past two years; 3) supply documented evidence that an effective system of surveillance is in operation in the FMD free zone where vaccination is practised as well as the buffer zone if applicable, that routine vaccination is carried out for the purpose of the prevention of FMD, and that the vaccine used complies with the OIE standards; 4) describe in detail: a) the boundaries of the FMD free zone where vaccination is practised and the buffer zone if applicable; b) the system for preventing the entry of infection into the FMD free zone; and supply evidence that these are properly supervised, and that all regulatory measures for the prevention and control of FMD have been implemented; 5) have a system of intensive and frequent surveillance for detection of any viral activity in the FMD free zone where vaccination is practised. The name of the free zone will be included in the list of FMD free zones where vaccination is practised only after acceptance of submitted evidence by the OIE. If a country that has "an FMD free zone with vaccination" wishes to change the status of the zone to "FMD free without vaccination", a waiting period of 12 months after vaccination has ceased is required. FMD infected countries Requirements for acceptance as an FMD free country are not fulfilled. FMD infected zone An FMD infected zone is a zone where the infection is present in a country with a free zone where vaccination either is or is not practised. The infected zone should be separated from the free zone either by a surveillance zone, or a buffer zone, or by physical or geographical barriers and animal health measures which effectively prevent the escape of infection. Live animals from FMD susceptible species can only leave the infected zone if moved by mechanical transport to the nearest designated abattoir located in the buffer zone or the surveillance zone for immediate slaughter. In the absence of an abattoir in the buffer zone or the surveillance zone, live FMD susceptible animals can be transported to the nearest abattoir in a free zone for immediate slaughter only under the following conditions: 1) no animals in the establishment of origin have shown clinical signs of FMD for at least 30 days prior to movement; 2) the animals were kept in the establishment of origin for at least three months prior to movement; 3) FMD has not occurred within a ten-km radius of the establishment of origin for at least three months prior to movement; 4) the animals must be transported under the supervision of the Veterinary Authority in a vehicle, which was cleansed and disinfected before loading, directly from the establishment of origin to the abattoir without coming into contact with other susceptible animals; 5) such an abattoir is not export approved; 6) all products obtained from the animals must be considered infected and treated in such a way as to destroy any residual virus. In particular, meat must be processed in accordance with Appendix 4.3.2.1.; 7) vehicles and the abattoir must be subjected to thorough cleansing and disinfection immediately after use. Animals moved into a free zone for other purposes must be taken to a quarantine station under the supervision of the Veterinary Authority. Freedom of infection of these animals must be established by appropriate tests. 54 Outbreaks in previously free countries or zones When FMD occurs in an FMD free country or zone where vaccination is not practised, the following waiting period is required to regain the disease free status: a) three months after the last case where stamping-out and serological surveillance are applied; or b) three months after the slaughtering of the last vaccinated animal where stamping-out, serological surveillance and emergency vaccination are applied. When FMD occurs in an FMD free country or zone where vaccination is practised, the following waiting period is required to regain the disease free status: a) twelve months after the last case where stamping-out is applied, or b) two years after the last case without stamping-out, provided that an effective surveillance has been carried out. Article 2.1.1.8. When importing from FMD free countries or zones where vaccination is not practised, Veterinary Administrations should require: for frozen semen of domestic ruminants and pigs the presentation of an international animal health certificate attesting that: 1) the donor animals showed no clinical sign of FMD on the day of collection and for the following 30 days; 2) the animals were kept in an FMD free country or zone where vaccination is not practised for at least three months prior to collection; 3) the semen was collected, processed and stored strictly in accordance with either Appendices 4.2.1.1. and 4.2.1.2. or Appendix 4.2.2.1., as relevant. Article 2.1.1.9. When importing from FMD free countries or zones where vaccination is practised, Veterinary Administrations should require: for semen of domestic ruminants and pigs the presentation of an international animal health certificate attesting that: 1) the donor animals: a) showed no clinical sign of FMD on the day of collection and for the following 30 days; b) were kept in a country or zone free from FMD for at least three months prior to collection; c) if destined to an FMD free country or zone where vaccination is not practised: i) have not been vaccinated and showed a negative response to tests for antibodies against FMD virus; or ii) had been vaccinated at least twice, with the last vaccination not more than twelve and not less than one month prior to collection; 2) no other animal present in the AI centre has been vaccinated within the month prior to collection; 3) the semen: a) was collected, processed and stored strictly in accordance with either Appendices 4.2.1.1. and 4.2.1.2. or Appendix 4.2.2.1; b) was stored in a country free from FMD for a period of at least one month between collection and export, and during this period no animals on the establishment where the donor animals were kept showed any sign of FMD. Article 2.1.1.10. When importing from FMD infected countries or zones, Veterinary Administrations should require: for semen of domestic ruminants and pigs the presentation of an international animal health certificate attesting that: 1) the donor animals: a) showed no clinical sign of FMD on the day of collection; b) were kept in an establishment where no animals had been added in the 30 days before collection and that FMD has not occurred within ten km for the 30 days before and after collection; c) have not been vaccinated and showed a negative response to tests for antibodies against FMD virus; or 55 d) 2) 3) had been vaccinated at least twice, with the last vaccination not more than twelve and not less than one month prior to collection; no other animal present in the AI centre has been vaccinated within the month prior to collection; the semen: a) was collected, processed and stored strictly in accordance with either Appendices 4.2.1.1. and 4.2.1.2. or Appendix 4.2.2.1.; b) was submitted, with negative results, to a virus isolation test if the donor animal has been vaccinated within the 12 months prior to collection; c) was stored for a period of at least one month between collection and export, and during this period no animals on the establishment where the donor animals were kept showed any sign of FMD. VESICULAR STOMATITIS Article 2.1.2.2. VS free country A country may be considered free from VS when: 1) VS is notifiable in the country; 2) no clinical, epidemiological or other evidence of VS has been found during the past two years. BLUETONGUE Preamble: Standards for diagnostic tests are described in the Manual. Article 2.1.9.1. For the purposes of this Code, the infective period for bluetongue virus (BTV) shall be 60 days (under study). The global BTV distribution historically has been shown to be between latitudes of approximately 40°N and 35°S. The BTV status of a country or zone within this part of the world can only be determined by a surveillance and monitoring programme (carried out in conformity with the provisions of Chapter 1.4.5.) using a statistically sound sample of BTV susceptible cattle (the whole sentence under study). The programme should provide at least a 95% level of confidence of detecting a seroconversion incidence of 2%. The BTV status of zones located outside this part of the world but adjacent to a zone within this part of the world which is not free should be similarly assessed (the whole sentence under study). Article 2.1.9.2. For the purposes of this Code: BTV free country or zone A country or a zone may be considered free from BTV when bluetongue is notifiable in the whole country and either: 1) the country or zone lies wholly north of 40°N or south of 35°S, and is not adjacent to a country or zone which is not free; or 2) a surveillance and monitoring programme has demonstrated no evidence of BTV in the country or zone during the past 2 years, nor have any ruminants been vaccinated against bluetongue in the country or zone during the past 12 months (under study); or 3) a surveillance and monitoring programme has demonstrated no evidence of Culicoides in the country or zone (under study). A BTV free country or zone in which surveillance and monitoring has found no evidence that BTV vectors are present will not lose its free status through the importation of seropositive or infective animals, or semen or embryos/ova from infected countries or zones. BTV seasonally free zone A BTV seasonally free zone is a part of an infected country or zone for which for part of a year, surveillance 56 and monitoring has demonstrated no evidence of BTV. For the application of Articles 2.1.9.5., 2.1.9.8. and 2.1.9.12., the seasonally free period is taken to commence the day following the last evidence of BTV transmission (as demonstrated by the surveillance and monitoring programme), and to conclude at least 28 days before the earliest date that historical data show bluetongue virus activity has recommenced (the whole sentence under study). The absence or scarcity of Culicoides during this period would provide an additional indication of seasonal freedom. A BTV seasonally free zone in which surveillance and monitoring has found no evidence that BTV vectors are present will not lose its free status through the importation of seropositive or infective animals, or semen or embryos/ova from infected countries or zones. BTV infected country or zone A BTV infected country or zone is a clearly defined area where evidence of BTV has been reported during the past 2 years. The infected country or zone should be separated from a free country or zone by a surveillance zone. Animals within the surveillance zone must be subjected to continuing surveillance. The boundaries of the surveillance zone must be clearly defined, and must take account of geographical and epidemiological factors that are relevant to BTV infection. Article 2.1.9.7. When importing from BTV free countries or zones, Veterinary Administrations should require: for semen of ruminants and other BTV susceptible herbivores the presentation of an international animal health certificate attesting that: 1) the donor animals: a) had been kept in a BTV free country or zone for at least 60 days (under study) before commencement of, and during, collection of the semen; or b) were subjected to a serological test to detect antibody to the BTV group, such as the BT competition ELISA or the BT AGID test, between 28 and 60 days (under study) after the last collection for this consignment, with negative results; or c) were subjected to a virus isolation test or polymerase chain reaction test (PCR) on blood samples collected at commencement and conclusion of, and at least every 7 days (virus isolation test) or at least every 28 days (PCR) during, semen collection for this consignment, with negative results; AND 2) the semen was collected, processed and stored in conformity with the provisions of either Appendices 4.2.1.1. and 4.2.1.2. or Appendix 4.2.2.2. Article 2.1.9.8. When importing from BTV seasonally free zones, Veterinary Administrations should require: for semen of ruminants and other BTV susceptible herbivores the presentation of an international animal health certificate attesting that: 1) the donor animals a) were kept during the BTV seasonally free period in a seasonally free zone for at least 60 days (under study) before commencement of, and during, collection of the semen; or b) were subjected to a serological test to detect antibody to the BTV group such as the BT competition ELISA or the BT AGID test, with negative results, at least every 60 days (under study) throughout the collection period and between 28 and 60 days (under study) after the final collection for this consignment; or c) were subjected to a virus isolation test or polymerase chain reaction test (PCR), with negative results, on blood samples collected at commencement and conclusion of, and at least every 7 days (virus isolation test) or at least every 28 days (PCR) during, semen collection for this consignment; AND 2) the semen was collected, processed and stored in conformity with the provisions of either Appendices 4.2.1.1. and 4.2.1.2. or Appendix 4.2.2.2. Article 2.1.9.9. When importing from BTV infected countries or zones, Veterinary Administrations should require: for semen of ruminants and other BTV susceptible herbivores 57 the presentation of an international animal health certificate attesting that: 1) the donor animals a) were kept in a Culicoides-proof quarantine station for at least 60 days (under study) before commencement of, and during, collection of the semen; or b) were subjected to a serological test to detect antibody to the BTV group such as the BT competition ELISA or the BT AGID test, with negative results at least every 60 days (under study) throughout the collection period and between 28 and 60 days (under study) after the final collection for this consignment; or c) were subjected to a virus isolation test or polymerase chain reaction test (PCR) on blood samples collected at commencement and conclusion of, and at least every 7 days (virus isolation test) or at least every 28 days (PCR) during, semen collection for this consignment, with negative results; AND 2) the semen was collected, processed and stored in conformity with the provisions of either Appendices 4.2.1.1. and 4.2.1.2. or Appendix 4.2.2.2. PARATUBERCULOSIS Article 3.1.6.1. Veterinary Administrations of importing countries should require: for domestic ruminants for breeding or rearing the presentation of an international animal health certificate attesting that the animals: 1) showed no clinical sign of paratuberculosis on the day of shipment; 2) were kept in a herd in which no clinical sign of paratuberculosis was officially reported during the 5 years prior to shipment; 3) showed negative results to diagnostic tests for paratuberculosis during the 30 days prior to shipment. BOVINE BRUCELLOSIS Article 3.2.1.1. Country or part of the territory of a country free from bovine brucellosis To qualify as free from bovine brucellosis, a country or part of the territory of a country shall satisfy the following requirements: 1) bovine brucellosis or any suspicion thereof is compulsorily notifiable in the country; 2) the entire cattle population of a country or part of the territory of a country is under official veterinary control and it has been ascertained that the rate of brucellosis infection does not exceed 0.2% of the cattle herds in the country or area under consideration; 3) the serological tests for bovine brucellosis are periodically conducted in each herd, with or without the ring test; 4) no animal has been vaccinated against bovine brucellosis for at least the past three years; 5) all reactors are slaughtered; 6) animals introduced into a free country or part of the territory of a country shall only come from herds officially free from bovine brucellosis or from herds free from bovine brucellosis. This condition may be waived for animals which have not been vaccinated and which, prior to entry into the herd, were isolated and were subjected to the serological tests for bovine brucellosis with negative results on two occasions, with an interval of 30 days between each test. These tests are not considered valid in female animals which have calved during the past 14 days. In a country where all herds of cattle have qualified as officially free from bovine brucellosis and where no reactor has been found for the past five years, the system for further control may be decided by the country concerned. Herd officially free from bovine brucellosis To qualify as officially free from bovine brucellosis, a herd of cattle shall satisfy the following requirements: 1) be under official veterinary control; 2) contain no animal which has been vaccinated against bovine brucellosis during at least the past three years; 58 3) 4) 5) only contain animals which have not showed evidence of bovine brucellosis infection during the past six months, all suspect cases (such as animals which have prematurely calved) having been subjected to the necessary laboratory investigations; all cattle over the age of one year (except castrated males) were subjected to serological tests with negative results performed twice at an interval of 12 months. This requirement is maintained even if the entire herd is normally tested every year or testing is conducted in accordance with other requirements established by the Veterinary Administration of the country concerned; additions to the herd shall only come from herds officially free from bovine brucellosis. This condition may be waived for animals which have not been vaccinated, come from a herd free from bovine brucellosis, provided negative results were shown following a buffered Brucella antigen test and the complement fixation test during the 30 days prior to entry into the herd. Any recently calved or calving animal should be retested after 14 days, as tests are not considered valid in female animals which have calved during the past 14 days. Herd free from bovine brucellosis To qualify as free from bovine brucellosis, a herd of cattle shall satisfy the following requirements: 1) be under official veterinary control; 2) be subjected to either a vaccination or a non-vaccination regime; 3) if a live vaccine is used in female cattle, vaccination must be carried out between three and six months of age, in which case these female cattle must be identified with a permanent mark; 4) all cattle over the age of one year are controlled as provided in paragraph 4) of the definition of a herd of cattle officially free from bovine brucellosis; however, cattle under 30 months of age which have been vaccinated using a live vaccine before reaching six months of age, may be subjected to a buffered Brucella antigen test with a positive result, with the complement fixation test giving a negative result; 5) all cattle introduced into the herd come from a herd officially free from bovine brucellosis or from a herd free from bovine brucellosis, or from a country or part of the territory of a country free from bovine brucellosis. This condition may be waived for animals which have been isolated and which, prior to entry into the herd, were subjected to the serological tests for bovine brucellosis with negative results on two occasions, with an interval of 30 days between each test. These tests are not considered valid in female animals which have calved during the past 14 days. Article 3.2.1.4. Veterinary Administrations of importing countries should require: for semen the presentation of an international animal health certificate attesting that: 1) when the semen is from an AI centre, the testing programme includes the serum-agglutination and complement fixation tests; 2) when the semen is not from an AI centre, the donor animals: a) were kept in a country or part of the territory of a country free from bovine brucellosis; or b) were kept in a herd officially free from bovine brucellosis, showed no clinical sign of bovine brucellosis on the day of collection and were subjected to a buffered Brucella antigen test with negative results during the 30 days prior to collection; or c) were kept in a herd free from bovine brucellosis, showed no clinical sign of bovine brucellosis on the day of collection and were subjected to the buffered Brucella antigen and complement fixation tests with negative results during the 30 days prior to collection; or d) showed no clinical sign of bovine brucellosis on the day of collection, were subjected to the buffered Brucella antigen and complement fixation tests with negative results during the 30 days prior to collection and no Brucella agglutinin was detected in the semen; 3) the semen was collected, processed and stored strictly in accordance with Appendices 4.2.1.1. and 4.2.1.2. as relevant. BOVINE TUBERCULOSIS Article 3.2.3.1. Country or part of the territory of a country officially free from bovine tuberculosis To qualify as officially free from bovine tuberculosis, a country or part of the territory of a country shall satisfy the following requirements: 59 1) 2) 3) 4) bovine tuberculosis is compulsorily notifiable in the country; 99.8% of the herds in the considered geographical area have been officially free from bovine tuberculosis for at least the past three years as disclosed by periodic testing of all cattle in the area to determine the absence of bovine tuberculosis (periodic testing of all cattle is not required in an area where a surveillance programme as described in paragraph 4) below, reveals that at least 99.9% of the cattle have been in officially tuberculosis-free herds for at least six years); cattle introduced into a country or part of the territory of a country officially free from bovine tuberculosis must be accompanied by a certificate from an Official Veterinarian attesting their compliance with Article 3.2.3.9. or the criteria set out in this Article; a country or part of the territory of a country officially free from bovine tuberculosis must have a Veterinary Administration which should be able to trace and test the herd of origin of any reactor to a tuberculin test disclosed after removal from the considered territory. Also animals which at a postmortem examination carried out by a veterinarian in an abattoir or elsewhere disclosed gross pathological lesions of tuberculosis which where necessary can be confirmed by established methods of microscopical-biological or cultural examination. In addition, such a country or part of the territory of a country officially free from bovine tuberculosis must have in place a surveillance programme to ensure the discovery of bovine tuberculosis should the disease be present in the country or part of the territory of a country, through slaughter monitoring and/or tuberculin testing. Herd officially free from bovine tuberculosis To qualify as officially free from bovine tuberculosis, a herd of cattle shall satisfy the following requirements: 1) the herd is in a country or part of the territory of a country officially free from bovine tuberculosis; or 2) all cattle in the herd: a) show no clinical sign of bovine tuberculosis; b) over six weeks of age, have shown a negative result to at least two official tuberculin tests carried out at an interval of six months, the first test being at six months following the eradication of bovine tuberculosis from the herd; c) showed a negative result to an annual tuberculin test to ensure the continuing absence of bovine tuberculosis; 3) cattle introduced into the herd: a) have been certified by an Official Veterinarian as having shown a negative result to the tuberculin test during the 30 days prior to entry into the herd; and/or b) were kept in a herd officially free from bovine tuberculosis. Article 3.2.3.7. Veterinary Administrations of importing countries should require: for semen the presentation of an international animal health certificate attesting that: 1) the donor animals: a) showed no clinical sign of bovine tuberculosis on the day of collection; b) were isolated in the establishment of origin during the three months prior to collection and were subjected to a tuberculin test for bovine tuberculosis with negative results on two occasions, with an interval of not less than 60 days between each test; or c) were kept in the exporting country for the 30 days prior to collection, in an establishment or AI centre where all animals are officially free from bovine tuberculosis; 2) the semen was collected, processed and stored strictly in accordance with Appendices 4.2.1.1. and 4.2.1.2. as relevant. ENZOOTIC BOVINE LEUCOSIS Article 3.2.4.2. EBL free herd 1) Qualification To qualify as free from EBL, a herd must satisfy the following requirements: a) there has been no evidence of EBL either clinical, post mortem, or as a result of a diagnostic test for EBL within the previous two years; 60 b) 2) 3) all animals over 24 months of age have been subjected to a diagnostic test for EBL on two occasions with negative results, at an interval of not less than 4 months during the preceding 12 months; c) animals introduced into the herd after the first test have fulfilled the conditions of Article 3.2.4.3.; d) all bovine semen and embryos/ova introduced into the herd after the first test have fulfilled the conditions referred to in Article 3.2.4.4. and Article 3.2.4.5. respectively. Maintenance of free status For a herd to maintain its EBL free status, the animals in the herd over 24 months of age on the day of sampling must be subjected to a diagnostic test for EBL with negative results at intervals of no more than 36 months and the conditions referred to in paragraphs 1) a), 1) c) and 1) d) above continue to be fulfilled. Suspension and restoration of free status If in an EBL free herd any animals react positively to a diagnostic test for EBL or a virological test (under study) for bovine leukosis virus, the status of the herd shall be suspended until the following measures have been taken: a) the animals which have reacted positively, and their progeny since the last negative test, must be removed from the herd immediately. However, any animal within the progeny which has been subjected to a PCR test with negative results (under study) may be retained in the herd; b) the remaining animals must have been subjected to a diagnostic test for EBL carried out as described in paragraph 1) b) above with negative results at least four months after removal of the positive animals and their progeny. Article 3.2.4.3. Veterinary Administrations of importing countries should require: for bovine semen the presentation of an international animal health certificate attesting that: 1) the donor bull was resident at the time of semen collection in an EBL free herd; and 2) if less than two years old, the bull came from a serologically negative "uterine" dam; or 3) the bull was subjected to diagnostic tests for EBL on blood samples on two occasions with negative results, the first test being carried out at least 30 days before and the second test at least 90 days after collection of semen; 4) the semen was collected, processed and stored strictly in accordance with Appendices 4.2.1.1. and 4.2.1.2. as relevant. INFECTIOUS BOVINE RHINOTRACHEITIS Article 3.2.5.2. Country or part of the territory of a country free from IBR 1) Qualification To qualify as free from IBR/IPV, a country or part of the territory of a country must satisfy the following requirements: a) the disease or suspicion of the disease is compulsorily notifiable; b) no animal has been vaccinated against IBR/IPV for at least three years; c) at least 99.8% of the herds are qualified as free from IBR/IPV. 2) Maintenance of free status For a country or part of the territory of a country to maintain its status free from IBR/IPV: a) a serological survey should be carried out annually on a random sample of the cattle population of the country or part of the territory of a country sufficient to provide a 99% level of confidence of detecting IBR/IPV if it is present at a prevalence rate exceeding 0.2% of the herds; b) all imported bovines comply with the provisions of Article 3.2.5.4.; c) all imported bovine semen and embryos/ova fulfil the requirements referred to in Article 3.2.5.6. or 3.2.5.7., and in Article 3.2.5.8. respectively. Article 3.2.5.3. IBR/IPV free herd 1) Qualification To qualify as free from IBR/IPV, a herd of cattle must satisfy the following requirements: 61 a) 2) all the animals in the herd have been subjected to a diagnostic test for IBR/IPV on a blood sample on two occasions with negative results, at an interval of not less than two months and not more than twelve months; or b) if the herd contains only dairy cattle of which at least a quarter are lactating cows, each of the latter has been subjected to a diagnostic test on individual milk samples carried out on three occasions at intervals of two months with negative results; c) animals introduced into the herd after the first tests referred to in paragraph a) or b) as relevant have been: i) kept in an IBR/IPV free herd; or ii) placed in isolation for a period of 30 days, and during this period have been subjected to a diagnostic test for IBR/IPV on a blood sample on two occasions with negative results, at an interval of not less than 21 days; d) all bovine semen and embryos/ova introduced in the herd after the first tests referred to in paragraph a) or b) as relevant have fulfilled the conditions provided in Article 3.2.5.6. or 3.2.5.7., and in Article 3.2.5.8. respectively. Maintenance of free status For a herd to maintain its status free from IBR/IPV, it must be subjected to the following tests with negative results: EITHER a) diagnostic tests for IBR/IPV on blood samples for all the animals repeated at maximum intervals of twelve months; in herds composed entirely of fattening animals, blood sampling may be limited to animals sent for slaughter; OR b) diagnostic tests on individual milk samples from all lactating cows repeated at intervals of six months. Veterinary Administrations applying an IBR/IPV eradication programme may extend these intervals (under study) if more than 98% of herds have been free from the disease for at least three years; and c) diagnostic tests on blood samples for IBR/IPV of all breeding bulls repeated at maximum intervals of twelve months; AND d) diagnostic tests on blood samples for IBR/IPV of all cattle having aborted after more than three months of gestation. Animals introduced into the herd must satisfy the conditions provided in paragraph 1) c) above, and semen and embryos/ova used in the herd must satisfy the conditions provided in Article 3.2.5.6. or 3.2.5.7., and in Article 3.2.5.8. respectively. Article 3.2.5.7. Veterinary Administrations of importing countries should require: for frozen semen the presentation of an international animal health certificate attesting that: 1) the donor animals were kept in an IBR/IPV free herd at the time of collection; or 2) the donor animals were held in isolation during the period of collection and for the 30 days following collection, and were subjected to a diagnostic test for IBR/IPV on a blood sample taken at least 21 days after collection of the semen, with negative results; or 3) if the serological status of the bull is unknown or if the bull is serologically positive, an aliquot of each semen collection was subjected to a virus isolation test, with negative results; and 4) the semen was collected, processed and stored strictly in accordance with Appendices 4.2.1.1. and 4.2.1.2. as relevant. HAEMORRHAGIC SEPTICAEMIA Article 3.2.12.1. For the purposes of this Code, haemorrhagic septicaemia (HS) is defined as a highly fatal disease in cattle and buffaloes caused by specific serotypes of Pasteurella multocida designated as 6:B and 6:E. The incubation period for the disease shall be 90 days (active and latent carriers occur). 62 Article 3.2.12.2. For the purposes of this Code: HS free country A country may be considered free from HS when: 1) the disease is compulsorily notifiable in the country; 2) no case of HS has occurred during the past three years. This period shall be six months after the occurrence of the last case for countries in which a stamping-out policy is practised, with or without vaccination against HS. Article 3.2.12.4. When importing from HS free countries or free zones, Veterinary Administrations should require: for cattle and buffaloes the presentation of an international animal health certificate attesting that the animals: 1) showed no clinical sign of HS on the day of shipment; and 2) were kept in a country or zone free from HS for at least six months or since birth. 63 Attachment 3. BOVINE SEMEN: OIE SANITARY CONTROL CONDITIONS 4.2.1. ARTIFICIAL INSEMINAT ION CENTRES Accreditation for export APPENDIX 4.2.1.1. BOVINE SEMEN A. AIMS OF CONTROL The purpose of official sanitary control in semen production is to maintain the health of animals on an artificial insemination (AI) centre at a standard which permits the international distribution of semen free of specific pathogenic organisms which can be carried in semen and cause infection in recipient cows or heifers. The disease position in one country generally differs from that in another, thus prophylactic programmes vary widely in the range of organisms for which donor bulls and teaser animals are tested before admission to an AI centre, while in isolation, and periodically after full admission into the stud. B. GENERAL CONDITIONS The designation of an AI Centre as 'accredited' and eligible to be used for the export of semen should be conditional on the fulfilment of certain requirements under official control. 1. 2. Artificial insemination centre a) The centre should be officially approved by the Veterinary Administration. b) The centre should be under the direct supervision and sanitary control of an Official Veterinarian. c) The centre should be under the overall supervision of the Veterinary Administration, which is responsible for routine visits to check the health and welfare of animals, and the procedures and prescribed records at the centre at least every six months. d) Only bovines associated with semen production should be permitted to enter the centre. Other species of livestock may exceptionally be resident on the centre provided they are kept physically apart from the bovines. e) Bovines on the centre should be adequately isolated from farm livestock on adjacent land or buildings for instance by natural or artificial means. f) The entry of visitors should be strictly controlled and personnel at a centre should be technically competent and observe high standards of personal hygiene to preclude the introduction of pathogenic organisms. Protective clothing and footwear for use only on the centre should be provided. g) Individual semen containers and storage rooms should be capable of being disinfected. Bulls and teaser animals 64 a) Bovines should only enter an AI centre if they fulfil the requirements laid down by the Veterinary Administration. b) The semen from bulls with genetical defects or associated with genetical defects in near relatives may not be eligible for export. c) Bovines must be clinically healthy and physiologically normal and must pass pre-entry tests within the 30 days prior to entry into isolation at an AI centre. The prescribed diseases and tests are listed in paragraph B.3.b). In addition to these tests, they must have undergone, with a negative result, a test on a blood sample for the isolation of bovine virus diarrhoea virus. This test shall be carried out on all animals over six months of age. 3. d) Bovines must remain in isolation at an AI centre for a period of at least 30 days before being retested to meet the standards listed in paragraph B.3. Bovines may only enter the stud on the successful completion of these tests and must be clinically healthy. e) The re-test requirements set out in paragraph d) may be dispensed with in countries (officially) free from bovine tuberculosis and bovine brucellosis. Also where trichomoniasis is not recorded and bovines only enter after sheath lavage as described in sub-paragraph iii) of B.3.b). Testing programme for bovines on AI centres a) Definitions Prescribed tests cover a minimal range of diseases from which all bovines on an AI centre must be free. Routine tests are tests applied at regular intervals to confirm the continued freedom from disease of the stud. b) Prescribed and routine tests i) Bovine tuberculosis Bovines to give negative results to intradermal tuberculin tests with mammalian tuberculin in accordance with Chapter 3.2.3. of this Code. In countries using an intradermal comparative test in a national disease control programme against tuberculosis, the requirement for a negative reaction to the mammalian tuberculin test may be waived. Routine tests should be applied at least every 12 months. ii) Bovine brucellosis Bovines to give negative results in accordance with Chapter 3.2.1. of this Code for B. abortus. Routine tests to be applied at least every 12 months. iii) Campylobacter fetus var. venerealis Bovines to meet the criteria set out in Chapter 3.2.2. of this Code; or alternatively, to have been subjected to a programme of sheath lavage with antibiotic solution of suitable strength and composition to eliminate Campylobacter infection. Routine tests or the programme of sheath lavage to be applied at least every 12 months. iv) Trichomoniasis 65 Bovines to meet the criteria set out in Article 3.2.6.2. of this Code. C. OPTIONAL TESTS AND REQUIREMENTS AI centres may be required by the Veterinary Administration to include in their veterinary prophylactic programmes a number of other diseases, either through vaccination or by requiring negative results to serological tests. Additionally, some importing countries may require assurances of freedom from a disease based on negative serology or other biological tests. The range of infections to be covered is extensive and beyond the capacity of AI centres to support totally. Thus, only optional tests can remain to be applied and interpreted by bilateral agreement when importation of semen is being considered. Where a disease is covered by a Chapter in this Code, the testing requirements of the Chapter should be followed. D. DILUENTS Whenever milk, egg yolk or any other animal protein is used in preparing the semen diluent, the product must be free of pathogens or sterilised; milk heat-treated at 92°C for 3-5 minutes, eggs from SPF flocks when available. The inclusion of penicillin, streptomycin, polymixin etc. is permitted provided this is declared in the international animal health certificate. E. SEMEN Semen for export should be stored separately in fresh liquid nitrogen in sterilised flasks for at least 28 days. The testing of ejaculates, and the dilution and freezing of semen must be carried out in a laboratory practising the hygienic standard set by the Veterinary Administration. Only semen of a health standard equivalent to that produced in an AI centre can be handled. Semen straws shall be code marked in line with national standards. Containers must be sealed before export and accompanied by an international animal health certificate listing the contents etc. F. DONOR BULL Records of the progeny of a donor bull should be maintained as far as possible to determine that it is not associated with any genetical defect. The records of the bull should indicate its fertility. The semen must be obtained from a bull with a normal libido. 66 4.2.1. ARTIFICIAL INSEMINATION CENTRES Accreditation for export APPENDIX 4.2.1.2. HYGIENIC COLLECTION AND HANDLING OF FRESH AND PRESERVED BOVINE SEMEN A. PURPOSE 1. Observation of the rules described below should result in the production of semen almost (though not necessarily entirely) free from common bacteria. 2. The population of common bacteria comprises representatives of the ubiquitous microflora, usually not pathogenic, which may be present in preserved semen. The bacteria encountered most frequently are saprophytic representatives of genera such as Micrococcus, Staphylococcus, Proteus, Bacillus and Corynebacterium, including some species which are occasionally pathogenic, such as Actinomyces pyogenes bovis (ex Corynebacterium pyogenes bovis), Staphylococcus aureus, streptococci of Lancefield groups A and D, Escherichia coli and Pseudomonas aeruginosa. 3. These rules shall apply to bulls kept in Artificial Insemination Centres under official supervision, which implies adoption of the OIE recommendations referred to in Appendix 4.2.1.1. 4. They also apply to individual bulls which are entirely free from genital diseases, even common ones, which may affect semen by the existence of an inflammatory reaction (presence of polymorphonuclear leukocytes in the ejaculate) and the presence of higher than normal concentrations of the immunoglobulins IgG1 and/or IgG2. 5. The recommended objective, i.e. to be aware of the presence of a limited population of common bacteria, and not necessarily complete freedom from bacteria, arises from the two following considerations: 6. a) There is no scientific information which demonstrates a significant correlation between the size of the population of common bacteria and expectations of fertility. The latter does not seem to be affected by the abundance of this population. b) Investigations of cows, whether during oestrus, or during the luteal phase on the occasion of embryo transfer, have revealed a population of common bacteria in the cervix and uterus, particularly in multiparous cows, qualitatively and quantitatively similar to that found in the male prepuce. However, the introduction of large numbers or particular combinations of such an exogenous microflora may weaken immune defences, leading to an infectious process. Finally, failure to observe these recommendations would contribute to an excessive contamination by this type of microflora. A bacterial count is consequently an excellent indicator of the competence of those responsible for implementing the hygienic rules in a bull stud, and for collecting and processing the semen samples. 67 B. MANAGEMENT OF BULLS The objective is the daily care of bulls to ensure a satisfactory state of cleanliness, particularly of the lower and ventral parts of the chest. 1. The bull should be kept under hygienic conditions at pasture, or if this is not possible in tethered or loose housing. If kept tethered, the litter must be kept clean and renewed as often as necessary. 2. The coat of the bull should be kept clean and generally short. 3. The length of the tuft of hairs at the preputial orifice, which is invariably soiled, should be cut to about 2 cm. The hair should not be removed altogether, because of its protective role. If cut too short, it may set up an irritation of the preputial mucosa. 4. The animal should be brushed regularly, and where necessary on the day before semen collection, paying special attention to the underside of the abdomen. 5. In the event of obvious soiling, there should be careful cleansing, with soap or a detergent, of the preputial orifice and the adjoining areas, followed by thorough rinsing and drying off. 6. In the case of an abnormally large preputial orifice or abnormalities within the cavity which might be accompanied by an invasion of micro-organisms, the preputial sac may be washed out before semen collection. Sterile saline solution is introduced several times into the prepuce using a catheter attached to a siphon tube. This precaution is vital if the subsequent ejaculate is to be tested for any pathogenic bacteria which might be present. 7. When the bull is brought out of its stall into the collection room, the technician must make sure that the bull is clean, and that it is not carrying any litter or particles of feed on its body or its hooves, for such materials are always heavily contaminated. C. SEMEN COLLECTION 1. The floor of the mounting area should be easy to clean, to dry and to disinfect. A dusty floor should be avoided. 2. The hindquarters of the teaser, whether a dummy or a live teaser animal, must be kept clean. A dummy must be cleaned completely after each period of collection. A teaser animal must have its hindquarters cleaned carefully before each collecting session. It is advisable to repeat the cleansing upon each change of bull, particularly in the case of soiling by defaecation. Plastic covers are poorly accepted by bulls, and are not generally used in practice. 3. The hand of the person collecting the semen must not come into contact with the bull's penis. The wearing of disposable, and preferably sterilised gloves is advisable to provide extra protection should the bull move unexpectedly. 4. It is necessary to clean the artificial vagina completely before each collection. It should have been dismantled beforehand, its various parts washed, rinsed and dried, and kept protected from dust. The inside of the body of the device and the cone should be sterilised before reassembly using approved sterilisation techniques to such as those involving the use of 70% ethyl or 98-99% isopropyl alcohol, ethylene oxide or steam. Once assembled it should be kept in a cupboard which is regularly cleaned and disinfected. 5. The lubricant used should be sterile and packed in tubes. The rod used to spread the lubricant must be sterile and should not be exposed to dust between successive collections. 6. It is recommended that the artificial vagina not be shaken after ejaculation, as otherwise lubricant and debris may pass down the cone to join the contents of the collecting tube. 68 7. When successive ejaculates are being collected, a new artificial vagina should be used for each` mounting. The vagina should also be changed when the bull has inserted its penis without ejaculating. 8. The collecting tubes must be sterile, and the recommended method of sterilisation is heating in an oven at 180°C for at least 30 minutes. They should be sealed while awaiting use, for example by a plug of sterile cotton wool, and kept in a sterile box or cupboard until required. 9. After collection, the tube should be left attached to the cone and within its sleeve until it has been removed from the collection room for transfer to the laboratory. D. HANDLING OF SEMEN AND PREPARATION OF SEMEN SAMPLES IN THE LABORATORY 1. 2. 3. Premises a) All laboratory operations should be carried out in an enclosed area, reserved exclusively for the purpose and not accessible to the stockmen on duty. b) The building should be well lit, with walls and floors easy to clean and disinfect, and provided with one or more benches which are also easy to clean and disinfect. c) It should be cleaned thoroughly each day, with removal of all dust. d) It is advisable to have a service hatch through which the semen sample can be passed, in order to reduce to a minimum the movement of air. e) The operations should be performed by personnel specifically allocated to the task, and in no case should they be performed by persons in charge of semen collection. Diluents a) All receptacles used should have been sterilised. b) Buffer solutions employed in diluents prepared on the premises should be sterilised by filtration (0.22 µm) or by autoclaving (121°C for 30 minutes) before adding egg yolk (or other non-egg yolk diluents) and antibiotics. c) If the constituents of a diluent are supplied in commercially-available powder form, the water used must have been distilled or demineralised, sterilised (121°C for 30 minutes), stored correctly and allowed to cool before use. d) When egg yolk or other egg derived diluents are used they should be added directly from the egg after puncture of the vitelline membrane. e) The diluent, which must never be prepared more than 72 hours before use, should be stored at +5°C in a stoppered flask. f) The addition of antibiotics is recommended. Procedure for dilution and packing a) The tube containing freshly-collected semen should be sealed as soon as it arrives in the laboratory, and kept sealed. b) After dilution and during refrigeration, the semen should also be kept in a stoppered flask. c) During the course of filling receptacles for dispatch (such as insemination straws), the receptacles and disposable tubes should be used immediately after being unpacked. Materials for repeated use should be sterilised with alcohol, ethylene oxide, steam or other approved sterilisation techniques. 69 4. d) If automatic equipment is used, the stainless-steel nozzles tubing etc. for filling and aspiration should be cleaned and sterilised. e) If sealing powder being used care should be taken to avoid contamination of it. Insemination procedure When a semen straw is used, it is first thawed and then dried with a clean cloth. The inseminator holds it close to the seal, where there is a pocket of air. This is the point where the straw is opened by cutting off the end with a pair of scissors which was kept in a tube containing 70° alcohol ethyl or 98-99% isopropyl alcohol. E. METHOD OF COUNTING THE MICRO-ORGANISMS IN SEMEN 1. To measure the extent of contamination of a sample of semen by the common microflora, by a standardised method, it is important to observe a number of rules for handling samples within the microbiological laboratory. 2. These rules concern: 3. a) appropriate media for dilution and counting; b) thawing and diluting procedures; c) inoculation and incubation of the cultures; d) method of calculating the total count; e) mode of expressing the results. Recommendations for the technique of counting micro-organisms present in semen are as follows: a) Media i) - Dilution media This is composed of buffered peptone water made up as follows: peptone NaCl Na2HPO4. 12H2O KH2PO4 Distilled water to pH = 7.0 ± 0.2 (25°C) 10.0 g 85.5 mM 25.1 mM 11.0 mM 1,000 ml (5.0 g) (9.0 g) (1.5 g) This solution is placed in 10 ml screw-capped tubes each containing 3.6 or 9 ml of solution, then it is sterilised in the autoclave for 20 minutes at 121°C. ii) - Agar for counting Tryptic soy agar is prepared as follows: Bacto Tryptone (pancreatic digest of casein) Bacto Soytone (papaic digest of soyabean meal) NaCl Bacto Agar Anhydrous glucose Distilled water to pH = 7.0 ± 0.2 (25°C) 15.0 g 5.0g 85.5 mM 15.0 g 5.6 mM 1,000 ml (5.0 g) (1.0 g) 70 This is placed in 30 ml flasks and then sterilised in the autoclave for 20 minutes at 121°C. It is then cooled to 45°C before use. For use the following are added under sterile precautions: - 5-10% inactivated, sterile bovine serum (56/58°C for 30 minutes) - 5% sterile erythrocyte extract - 3 ml SPS (sodium polyanethol sulphate) 5% solution. iii) White or plain agar Prepared from 9-18 g agar-agar (according to source), made up to 1,000 ml with distilled water. b) Method of preparing dilutions of semen i) Thawing Prepare one tube containing 3.6 ml dilution media and four tubes containing 9 ml. The samples of semen are kept in liquid nitrogen until required. They are then thawed in a water bath at 37°C for 2 minutes before being transferred to cultures. Two semen samples from the same ejaculate are required for each analysis. ii) Dilution After thawing, rapidly dry the semen container then disinfect it with 70° ethyl or 98-99% isopropyl alcohol. After opening, transfer the two semen samples into a sterile tube. Measure exactly 0.4 ml of semen and place this in the tube containing 3.6 ml of dilution medium (1:10 dilution). Mix the contents by stirring (vortex apparatus), then prepare dilutions of 10-2 to 10-5 in four tubes each containing 9 ml of dilution medium (1 ml + 9 ml). c) Inoculation and incubation Take 0.5 ml of each dilution and introduce under sterile conditions into each of four Petri dishes, 910 cm diameter. Add to each Petri dash about 15 ml of counting agar cooled to 45°C. Mix by circular agitation. Allow to solidify on a level surface. In order to prevent invasion of the culture medium by certain saprophytic bacteria, the surface of the inoculated and cooled medium may be covered with 4 ml of white (plain) agar cooled to 45°C, if there is room. Incubate the dishes for 48-72 hours at 37°C. d) Counting and method of calculation After 72 hours of incubation at 37°C, the colonies are counted by the standard procedure, selecting for the purpose the dilution which contains between 30 and 300 colony-forming units per dish. The method of calculation is that specified in the current International Standard (ISO 4833). e) Expression of the results Record the number of colony-forming units counted. Multiply by the dilution factor. Express the results as the number of micro-organisms or colony-forming units (CFU) per sample or per ml of semen. 71
