Vol. 60 3) 2005
THE SAFETY AND EFFICACY OF THE IMMUNOTHERAPY
TREATMENT Y-COMPLEX
IN A CLINICAL MASTITIS STUDY UNDER FIELD
CONDITIONS
1 Pinchasov Y.¬ 1 Spanier J., 2 Lietner G., 1 Pitcovsky J., 1 Morag E., 1 Raymond D. and 1 Gordon A.
1. Yamit Biotechnologies Ltd. - B.O. Box 408, Kyriat Shmone, 11013 Israel
2. National Mastitis Reference Center, Kimron Veterinary Institute, P.O. Box 12,
Bet Dagan 50250, Israel
Introduction
Immunotherapy is a novel medical approach that is gaining growing interest
(oncology, rheumatology, viral diseases etc.), and can offer an alternative for conventional medications. Immunotherapy is defined as a process designed to stimulate the defensive immunological system in confronting foreign substances, such as bacteria and viruses, as pathogens.
Mastitis accounts for the majority of financial loss in dairy cattle and despite the numerous endeavors invested in prevention; hitherto most treatments resulted in partial success. Recently the awareness of healthy food has grown and became evident in the aim to minimize the usage of antibiotics among farms including mastitis in dairy cattle. The current state of affairs could be summarized as follows:
1. Partial success rates with overall disappointment from present medications;
Increasing the risk for selection of resistant bacterial strains as a result of excessive use of antibiotics;
3. Upsurge in the danger posed to public health due to antibacterial residues in the milk following treatment.
Hence, there emerges the need for expanding the arsenal of medical formulations available for the treatment of bovine mastitis (as well as other diseases), which result in better cure rates, reduced bacterial resistance and minimal, if any, harm to consumers.
The Y - complex is a biological, broad-spectrum antimicrobial agent based on specific antibodies activity combined with inducers comprising components of the immune system (Fig. 1). Currently, specific antibodies are directed against three types of bacteria: Escherichia coli, Streptococcus dysgalactiae and
Streptococcus uberis.
Figure 1 . Schematic figure of Y-Complex - Ig YComplex® Targeting Specific
Phagocytosis of a Target Agent

When administrated to an infected quarter, the mode of action could be described in a schematic way as follows:
1. Bacteria are bound to the antibodies on the surface of the carrier to create units of complex with pathogens. Several pathogens are bound to each carrier.
2. The inducers on the complex stimulate the phagocytes and attract them.
3. The phagocytes engulf the complex and digest the bound pathogens.
4. Pathogens are destroyed and removed from the udder during milking.
The Y-Complex was tested in various controlled experiments in cows to demonstrate its safety and efficacy. To establish whether it causes adverse effect in the udder, Y-Complex was intramammary administrated into healthy udders at different doses (up to 10-fold the recommended dose). The local reactions to the treatment were monitored, and except for a slight increase in somatic cell count (SCC) throughout the 24 hours following administration and a slight negligible temporary reduction in milk yield, no adverse systemic effects were observed.
Several preliminary challenge studies were performed with different pathogens (E. coli, Strep. dysgalactiae) in 12 cows (6 for each pathogen) at the Volcani Center (1).
Cows selected for the study were challenged in two quarters in parallel (either front or rear ones) with one of the pathogens mentioned. Following 6-8 hours (E. coli) or 3 days (Strep. dysgalactiae) one quarter was infused with Y-Complex, whereas the opposite quarter in each cow remained untreated. This experiment showed a significant improvement in the quarters treated with Y-Complex with both pathogens.
Cure was defined as: A) time of bacterial eradication; B) improvement in clinical

symptoms, and C) return to baseline levels of SCC prior to challenge.
The objective of the present study was to examine the safety and efficacy of the Y-
Complex compared with other medications (Standard of Care) in cows with spontaneous mastitis in field settings.
Materials and Methods
The study was conducted over three months between November 2003 and January
2004, at Kazir Va'Keshet site, on a herd of about 500 cows. The local facilities included outdoor open barns with straw bedding and the cows were fed with a total mixed ration. Milking was performed 3 times a day in a herring-bone type milking parlor. Average milk yield in 2003 was 11,244 liters per cow. For securing a proper and balanced assignment, the cows were a priori designated into two experimental groups (A, B), based on their lactation number days in milk, previous level of SCC and average milk yield. Once mastitis was detected, a cow was treated according to its pre-assigned group (in blocks): Treatment A: Y-Complex, or Treatment B: (Positive
Control) sulfarimethoprim. This enabled the examination of the efficacy of Y-
Complex® in more homogeneous experimental groups. The study investigated Y-
Complex® formula (Group A) contained 60 millions units given in single-use prefilled syringes containing 10 ml Y-Complex® preservative-free physiologically buffered saline solution. Treatment consisted of 2 intramammary pre-filled sterile administrations of 10 ml/quarter, each, with an interval of 18 to 24 hrs. The Y-
Complex® was given immediately after milking and the next milking was skipped to allow sufficient drug concentration in the infected quarter.
The milk of this group was discarded for three consecutive days according to instructions of the ethical committee for this study. The control group (B) was treated with the following combinations: Flunixin meglumine (NSAID) Ñ 200 mg/100 kg
BW IM, once a day for 2 d consecutively or Sulphadiazine + Trimethoprim when E. coli was suspected, 20 mg/ kg, once a day for 3 d consecutively, or Procaine Penicillin
6 mil IU + 7.5 gr Streptomycine / 500kg BW once a day for 3 d. Additional hand milking was conducted (Standard of Care), and oxytocin 20-50 and IU
Intramuscularly (selected cases only) Some cases were milked as an exclusive treatment.
Overall, 26 cows were reported with clinical mastitis (mostly with infection in one quarter, and some in two quarters). Three cows from the Y-Complex® group were excluded in the first 24 hours due to severe infection and the need to avoid risks to the animal welfare. A total of 23 cows completed the full course of the study; 10 in the Y-
Complex® group and 13 in the control group.
Prior to treatment and on days 1, 2, 7, 14 and 28 following administration, the infected udder appearance was clinically examined as well as changes in milk texture
(normal, small flakes, clots, serous with or without clots, hemorrhagic, CMT, SCC and bacteriological analysis. The sponsor’s qualified veterinarian conducted clinical examinations, while SCC and bacteriology were performed at Caesarea and Kimron
Institute (2,3). Backup bacteriological isolation was the responsibility of the reference laboratory at Kimron Institute. Clinical examination included rectal temperature, heart and respiratory rates, rumen activity and hydration status. Physical examination of the udder included: evaluation of the quarter and various milk characteristics (4), milk

yield (daily, mean of last 10 days) and milk conductivity were recorded. In addition, milk parameters and SCC prior to mastitis episode, up to three months thereafter, were also recorded. Resulting data were statistically analyzed using student's T-test.
Results
1.
Bacteriology: The proportion of positive bacterial isolations from initial tests
(day of infection appearance) was 45.5%, out of which E. coli 54%, and 46% were Strep. dysgalactiae and streptococci species group C. In over 50% of the samples no bacteria were identified. Thus it was assumed (according to clinical examination) that environmental gram-negative bacteria caused the infections. Of the total bacterial isolations, half were in the Y-Complex® group and the rest in the control group.
2.
Pretreatment clinical observations: Pathological changes in the infected udders (swelling, edema, stiffness) were observed among 70% of the cows in the Y-Complex® group and 54% in the control group. No significant differences were found in systemic reactions between the 2 groups. However, cows of the Y-Complex® group had higher heart rates and rumen activity.
3. Milk parameters: Changes in CMT, SCC and normal milk characteristics in the infected quarters were observed in all cows. CMT values exceeded 3 prior to treatment and declined steadily throughout the follow-up 28 days period, but remained relatively high (±2). No significant differences were found during this period between the groups in these parameters. Average daily milk yield was similar in both groups 10 days prior to infection. On the day of treatment, a reduction in milk yield was observed in approximately 25%-30% of all the cows (Table 1). Seven days following treatment the milk yield returned returned to the pre-infection level in the Y- Complex® group, whereas that level remained low in the control group. All cows returned to the pre-infection level at the end of the 28 days follow-up.
Table 1.
Average milk yield per cow and periodical differences ten days before treatment (-10), day of treatment (0), 7 and 28 days following treatment of cows with clinical mastitis treated either with Y-Complex® or Standard of
Care (control).
Milk yield differences were calculated based on daily figures, 10 days prior to infection.
Experimental Time (days)
-10
-1
0 (Treatment)
(
1
SOC)
Milk difference (L) Milk production (L)
-
-5.0
-11.7
36.8
31.8
25.1
Y- Complex
®
Milk difference (L)
-
-4.1
-13.0
Milk production (L)
36.1
32.0
23.1
P (F)
2
NS
NS
NS

7
28
-1.9
-1.0
1
SOC - Standard of Care
2
NS=P<0.05
34.9
37.8
-01.1_
-0.1
Seeing that the decline of SCC following clinical mastitis may take longer than that of the follow-up time, the cows' data were retrieved from the farm records (Herd Book) one month prior to the infection and up to three months post-treatment. Mean SCC figures are presented in figure 2.
Figure 2.
Mean SCC as recorded in the Herd Book prior to the infection (day 0), and 27, 68 and 99 days post-treatment of cows treated with Y-Complex® or SOC controls.
37.2
36.0
NS
NS
Note: Day 0 - SCC at 17/11/03 prior to infection; Day 99 - SCC at 20/2/04, post-treatment period.
Discussion and conclusions
The prime objective of the study was to examine the safety and efficacy of the YComplex® compared with Standard of Care comprising antibiotics and supportive anti-inflammatory treatment in cows with spontaneous mastitis.
The basic for the success of the treatment was:
Clinical cure, disappearance or relief in local or systemic clinical symptoms; functional recovery - return to original milk yield or at least an increase in yield

in comparison to the day of infection; bacterial cure - bacterial eradication; tissue recovery - significant reduction in SCC during the course of time that similar to observed following antibiotic treatment.
The premise was found correct, meaning that no significant difference was found between the two groups in the overall parameters examined during the course of this study.
One of the problems each farmer encounters is the lack of identification of the causative microorganism at the time of diagnosing the clinical symptoms of infection. Proper identification of the pathogen requires at least 24 hours
(from the arrival of the milk sample to the laboratory) and the establishment of sensitivity to specific antibiotic (antibiogram) may take another 24 hours. This is the major drawback in making a decision of which antibiotic to use.
Therefore most of the decisions to treat an infected quarter are based on prevalence of pathogens on the farm of previous mastitis episodes. The formulation of YComplex® used in this study was designed to treat the most prevalent pathogens known at the farm prior to the study. In fact, those pathogens were identified in only less than 50% of the mastitis episodes during the course of the study. Thus, it is recommended to consider better formulation of the YComplex® with broader antibacterial activity.
Episodes caused by gram-negative bacteria may last a few days. However, the recovery time in terms of milk yield and reduction in SCC may take up to a few months. In this study, recovery time among cows treated with Y-
Complex® was significantly shorter (99 days), returning to normal levels prior to infection (±100,000 cells), as opposed to cows in the control group that were treated with antibiotics with SCC with over 1,000,000 cells after three months post-treatment.
Y-
Complex® was found safe for intramammary administration. No major adverse events were observed except for a slight temporary reduction in milk yield and increased SCC due to a local reaction during the first 24 hours after treatment.
The results of previous studies and the present field study indicate that the Y-
Complex® is a friendly immunotherapy for both animal and consumer and is superior to antibiotics concerning public health. In the long run, the product poses two major advantages over antibiotics: minimize the chance for selecting of resistant and more virulent strains of microorganisms as a result of repeated use, and neutralizing the risk for consumers due to drug residues in the milk. Further to these advantages, there is an economical benefit to the farmer in the short run by continuation of marketing the milk from the untreated quarters. The basic calculation of milk loss from systemic antibiotic treatment till complete clearance of medication results in 5 days of milking.
According to milk records received in this study, each cow lost on average
125 liters (25 liters per day for five days) or 177 NIS as a result of milk discarding, whereas in the YComplex® group, milk was discarded from the infected quarter for two days only.

In summary, no significant differences were noted between the treatments with standard antibiotics or YComplex® during the first month of the mastitis episode. However, an advantage was found in the time of SCC returning to normal values after infection. The recovery in SCC along with the return to normal milk yield indicates functional and tissue cures. Furthermore, in farms where a problem of high SCC exists in tank milk, returning to normal SCC affects the entire herd rather than the individual cow. Minimizing the chance for medication residue in milk (inhibiting substances) and the ability to sell the milk during treatment and afterwards, portray the YComplex® as a good substitute for standard antibiotics in clinical mastitis. These are preliminary results due to the small number of cows in the single herd that participated in this study. Further studies are required to report the encouraging results of this study on a larger number of cows on different farms.
LINKS TO OTHER ARTICLES IN THIS ISSUE
References
1. Pinchasov Y., Spanier J., Gordon A., Pitcovsky J., Morag E., Lietner G.,
Tiomkin D., Braun R. Efficiency and safety of Y- complex in mastitis therapy:
Field trial.16th Congress on Ruminant Dairy & Milk Science, Israel, pp. 55-56,
2004.
2. Leitner G., E. Lubashevsky, A. Glickman, M. Winkler, A. Saran and Z.
Trainin. Development of a Staphylococcus aureus vaccine against mastitis in dairy cows. I. Challenge trials.J. Vet. Immunol. Immunopath., 93:31-38, 2003.
3. Leitner G., N. Yadlin, E. Lubashevsky, E. Ezra, A. Glickman, M. Chaffer, M.
Winkler, A. Saran and Z. Trainin. Development of a Staphylococcus aureus vaccine against mastitis in dairy cows. II. Field trials.J. Vet. Immunol.
Immunopath., 93:153-158, 2003.
4. Anderson K.L., Smith A.R., L., Shanks R. D., Davis L. D., Gustafsson B.K.
Efficacy of flunixin meglumine for the treatment of endotoxin-induced bovine mastitis. Am. J. Vet. Res. 1986: 1366-1372.
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