Research Article - Annals of Biological Research ( 2018) Volume 9, Issue 4
Present study is aimed to determine the excretion of Brucella abortus organisms in milk of vaccinated lactating cattle and buffaloes. All experimental lactating animals in early and mid-stage of lactation were vaccinated with Brucella abortus S19 reduced dose vaccine through subcutaneous route. After vaccination daily milk samples from all the animals i.e. 10 cattle and 10 buffaloes were collected in sterile sample collection vials from day 1 till day 30 for bacteriological analysis. Milk collected from all vaccinated animals was not used for human consumption and was discarded throughout the study period. Sterile Potato infusion agar (PIA) media plate was used for detection of Brucella abortus organisms. For identification of contaminants BDBBL Crystal ID (Becton, Dickinson and Company) was used. All the experimental lactating animals were serologically negative for brucellosis at the time of enrollment. Milk samples from the animals that were collected and tested up to 30 days, were found negative for Brucella abortus in microbiological culture, although in all of the milk samples, contamination was observed.
Bovine brucellosis, Brucella abortus S19 vaccine, Milk excretion
Bacterial organisms of genus Brucella that are pathogenic for wide variety of animals and humans beings. [1]. In animals, abortion is the most obvious manifestation. Infections may also cause stillbirths or weak calves, retained placentas and reduced milk yield [2]. Brucellosis is readily transmissible to humans, causing acute febrile illness and undulant fever which may progress to a more chronic form and can also produce serious complications affecting the musculo–skeletal, cardiovascular and central nervous systems. Infection is often due to occupational exposure and is essentially acquired by the oral, respiratory or conjunctival routes but ingestion of dairy products constitutes the main risk to the general public where the disease is endemic [3].
Public health importance of brucellosis is much related to the infected animal species from which human transmission occurs. The most rational approach for preventing human brucellosis is the control and elimination of the diseases in animal reservoir and health education of the public working at high risk area [4].
Brucellosis is an occupational hazard. It is primarily a disease of animals transmitted directly or indirectly to man. Dairy workers, shepherds, veterinarians, abattoir workers and animal husbandry personnel are particularly at risk. It constitutes an uncontrolled public health problem in many developing countries [5]. Infected cows shed Brucella in their milk and this is key in its transmission to calves. In dairies, milking is another mode of transmission that must be taken into account because the bacteria are highly likely to be transmitted from cow to cow if the same teat cups are used for milking [6].
The mammary gland and regional lymph nodes can also be infected and bacteria can be excreted in milk [7]. Female calves can also be infected during birth when passing through infected the birth canal or by suckling colostrum or milk from infected cows. While most of these calves rid themselves of Brucella a small percentage may continue to be infected until adulthood remaining negative to diagnostic serological tests but aborting during their first pregnancy. Such animals pose a serious threat to brucellosis control and eradication [8].
Brucella abortus S-19 has remained the cornerstone for most Brucellosis eradication program since 1940. Calfhood vaccination with S-19 vaccine lead to progressive reduction in cases of brucellosis globally. Vaccination is usually performed on young female calves between three to eight months of age. Vaccination of adult cattle with S19 low dosage (3 × 108 – 3 × 109) CFU was also successfully employed in infected herds. In general, after vaccination S19 calfhood does not persist in the reproductive tracts of mature heifers and does not cause abortion in these animals. Nonetheless, even with markedly infrequent occurrence, some cattle remain chronically infected and may abort and excrete vaccine strain in the milk. Almost all cows vaccinated with strain 19 do not shed the organisms in milk for any length of period. It is shed in milk for a day or two in a few animals (less than 1%).
The present study was undertaken to evaluate safety of Brucella abortus S-19 reduced dose (S/C) vaccine in lactating animals and to determine if any excretion of Brucella abortus organisms in milk occurs in vaccinated lactating animals.
10 lactating cattle and 10 lactating buffaloes, seronegative for Brucella antibodies, were enrolled in the study. The animals were at first and second stage of lactation at the time of vaccination with Brucella abortus S19 reduced dose subcutaneous vaccine (Tables 2 and 3). All the animals received 2 ml dose of 3 × 108 to 3 × 109 viable Brucella abortus S19 vaccine subcutaneously in the mid neck region. The animals were observed for excretion of Brucella abortus organisms in milk throughout the study period of 30 days. Daily milk samples from all the animals (10 cows + 10 buffaloes) were collected in sterile sample collection vials from day 1 till day 30 for bacteriological analysis. Aseptic milk collection was ensured by teat dip and udder spray with disinfectant solution before milk collection. Milk was not used for human consumption and was discarded from all vaccinated animals throughout the study period.
Sterile Potato infusion agar (PIA) media plate was used in this study [9,10]. One vial of Brucella selective supplement (suspension of 1 vial of Brucella selective supplement in 50% methanol) was added in 500 ml of Brucella media. The plates were observed for any colony formed at each dilution and if present, identified by Gram’s staining and observed for the morphology of Brucella under microscope.
BDBBL Crystal ID: (Becton, Dickinson and Company)
The isolated colony from the inoculum was suspended in the tube of BBL crystal respective inoculum fluid. As per the procedure of BDBBL crystal further it was incubated for 18-20 hours at 35-37oC for Enteric/Non Fermentor, 18-24 hours at 35-37°C for gram positive and 4.0 hours at 35-37°C for anaerobic organisms(Table 1).
| Number of lactating animals vaccinated | Stage of lactation |
|---|---|
| 4 | Early lactation (cattle) |
| 6 | Mid lactation (cattle) |
| 7 | Early lactation (buffaloes) |
| 3 | Mid lactation (buffaloes) |
Table 1: Stage of lactation of the enrolled animals at the time of Brucella abortus S19 reduced dose S/C vaccination.
All the lactating animals i.e. 10 cattle and 10 buffaloes were serologically negative for brucellosis at the enrollment. They were administered with Brucella abortus S19 reduced dose vaccine through subcutaneous route. Milk samples from all the experimental animals that were collected and tested up to 30 days. The milk sample test showed negative for Brucella abortus in microbiological culture, although in all the milk samples, contamination was observed. The details of contaminants observed in the milk of cattle and buffaloes is given in table 2 and 3 respectively.
| Bruvax Plus (Brucella abortus S19 reduced dose)-Milk excretion study-(Route of Administration:S/C wth 2ml) | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cow ( ILFC,College of Veterinary Science,Rajendranagar,Hyderabad) | |||||||||||||
| Stage of lactation | Early lactation | Mid lactation | Remarks | ||||||||||
| Animal Tag No: | 216 | 232 | 1333 | 1334 | 1339 | 1366 | 37 | 40 | 115 | 122 | |||
| S. No | Sample details | Day of collection | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | |
| 1 | Cow milk | Day1 | 5 | 3 | 3 | 4 | 4 | 6 | 6 | 3 | 4 | 3 | The following microbial contaminants observed 1) Brevundimonas diminuta 2) Kytococcus sedentarius 3) Agrobacterium tumifaciens 4)Strepto coccus vestibularis 5)Micrococcus luteus 6)Lactococcus rafinolactis . No Brucella organisms were found. |
| 2 | Cow milk | Day2 | 6 | 5 | 4 | 3 | 3 | 4 | 3 | 3 | 5 | 6 | |
| 3 | Cow milk | Day3 | 5 | 4 | 3 | 3 | 4 | 6 | 5 | 4 | 4 | 3 | |
| 4 | Cow milk | Day4 | 3 | 4 | 4 | 4 | 6 | 5 | 4 | 3 | 5 | 4 | |
| 5 | Cow milk | Day5 | 6 | 4 | 5 | 3 | 6 | 4 | 4 | 4 | 5 | 3 | |
| 6 | Cow milk | Day6 | 3 | 4 | 3 | 4 | 3 | 3 | 3 | 4 | 5 | 4 | |
| 7 | Cow milk | Day7 | 5 | 4 | 4 | 3 | 5 | 6 | 3 | 3 | 6 | 3 | |
| 8 | Cow milk | Day8 | 3 | 4 | 3 | 6 | 5 | 4 | 3 | 5 | 5 | 3 | |
| 9 | Cow milk | Day9 | 3 | 3 | 5 | 4 | 4 | 5 | 5 | 3 | 3 | 6 | |
| 10 | Cow milk | Day10 | 4 | 4 | 4 | 5 | 5 | 4 | 3 | 4 | 5 | 5 | |
| 11 | Cow milk | Day11 | 3 | 5 | 4 | 4 | 4 | 5 | 3 | 5 | 6 | 4 | |
| 12 | Cow milk | Day12 | 4 | 6 | 5 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | |
| 13 | Cow milk | Day13 | 5 | 5 | 6 | 3 | 6 | 3 | 3 | 5 | 4 | 5 | |
| 14 | Cow milk | Day14 | 6 | 4 | 5 | 4 | 6 | 4 | 3 | 4 | 5 | 3 | |
| 15 | Cow milk | Day15 | 5 | 4 | 4 | 3 | 3 | 3 | 5 | 3 | 6 | 3 | |
| 16 | Cow milk | Day16 | 3 | 3 | 5 | 4 | 3 | 3 | 5 | 4 | 3 | 6 | |
| 17 | Cow milk | Day17 | 5 | 3 | 4 | 6 | 5 | 3 | 3 | 5 | 3 | 4 | |
| 18 | Cow milk | Day18 | 4 | 3 | 3 | 3 | 4 | 5 | 3 | 4 | 5 | 4 | |
| 19 | Cow milk | Day19 | 6 | 4 | 6 | 4 | 3 | 5 | 6 | 3 | 3 | 3 | |
| 20 | Cow milk | Day20 | 5 | 5 | 3 | 5 | 4 | 3 | 6 | 5 | 3 | 3 | |
| 21 | Cow milk | Day21 | 6 | 3 | 5 | 4 | 5 | 4 | 6 | 4 | 5 | 3 | |
| 22 | Cow milk | Day22 | 6 | 3 | 4 | 4 | 4 | 3 | 6 | 5 | 3 | 6 | |
| 23 | Cow milk | Day23 | 6 | 4 | 3 | 4 | 5 | 3 | 4 | 4 | 4 | 4 | |
| 24 | Cow milk | Day24 | 3 | 3 | 6 | 5 | 4 | 3 | 6 | 5 | 4 | 6 | |
| 25 | Cow milk | Day25 | 4 | 6 | 5 | 5 | 5 | 3 | 4 | 3 | 4 | 3 | |
| 26 | Cow milk | Day26 | 5 | 5 | 3 | 5 | 3 | 6 | 4 | 4 | 4 | 3 | |
| 27 | Cow milk | Day27 | 4 | 4 | 6 | 5 | 4 | 3 | 5 | 3 | 5 | 3 | |
| 28 | Cow milk | Day28 | 5 | 4 | 5 | 3 | 5 | 3 | 4 | 6 | 5 | 3 | |
| 29 | Cow milk | Day29 | 4 | 5 | 3 | 5 | 3 | 6 | 6 | 4 | 5 | 3 | |
| 30 | Cow milk | Day30 | 5 | 3 | 5 | 6 | 5 | 5 | 3 | 6 | 4 | 5 | |
Table 2: Excretion of Brucella organisms and microbial contaminants identified in milk of cattle vaccinated with Brucella abortus S19 reduced dose S/C vaccine.
| Bruvax Plus (Brucella abortus S19 reduced dose) - Milk excretion study - (Route of Administration : S/C wth 2ml) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Buffalo ( ILFC,College of Veterinary Science,Rajendranagar,Hyderabad) | |||||||||||
| Stage of lactation | Early lactation | Mid lactation | |||||||||
| Animal Tag No: | 430 | 497 | 457 | 498 | 519 | 530 | 435 | 509 | 505 | ||
| S.No | Sample details | Day of collection | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count | Viable Count |
| 1 | Buffalo milk | Day1 | 4 | 3 | 5 | 3 | 6 | 3 | 4 | 6 | 4 |
| 2 | Buffalo milk | Day2 | 3 | 6 | 4 | 3 | 4 | 3 | 3 | 4 | 5 |
| 3 | Buffalo milk | Day3 | 5 | 6 | 3 | 5 | 3 | 4 | 3 | 6 | 3 |
| 4 | Buffalo milk | Day4 | 3 | 5 | 5 | 3 | 4 | 5 | 3 | 4 | 4 |
| 5 | Buffalo milk | Day5 | 6 | 3 | 4 | 3 | 5 | 3 | 4 | 3 | 4 |
| 6 | Buffalo milk | Day6 | 5 | 3 | 4 | 3 | 4 | 3 | 6 | 3 | 5 |
| 7 | Buffalo milk | Day7 | 3 | 6 | 3 | 5 | 6 | 4 | 3 | 4 | 5 |
| 8 | Buffalo milk | Day8 | 4 | 4 | 5 | 4 | 4 | 3 | 4 | 3 | 6 |
| 9 | Buffalo milk | Day9 | 6 | 3 | 5 | 4 | 3 | 5 | 3 | 3 | 4 |
| 10 | Buffalo milk | Day10 | 4 | 3 | 3 | 4 | 5 | 3 | 6 | 5 | 5 |
| 11 | Buffalo milk | Day11 | 3 | 4 | 3 | 5 | 6 | 3 | 5 | 4 | 5 |
| 12 | Buffalo milk | Day12 | 5 | 3 | 3 | 5 | 5 | 4 | 3 | 4 | 3 |
| 13 | Buffalo milk | Day13 | 3 | 4 | 3 | 4 | 3 | 3 | 4 | 5 | 3 |
| 14 | Buffalo milk | Day14 | 4 | 5 | 3 | 6 | 3 | 5 | 3 | 4 | 3 |
| 15 | Buffalo milk | Day15 | 4 | 3 | 4 | 3 | 3 | 4 | 3 | 5 | 5 |
| 16 | Buffalo milk | Day16 | 5 | 6 | 5 | 4 | 3 | 3 | 3 | 4 | 3 |
| 17 | Buffalo milk | Day17 | 3 | 4 | 5 | 4 | 4 | 5 | 6 | 3 | 4 |
| 18 | Buffalo milk | Day18 | 3 | 3 | 4 | 4 | 5 | 4 | 5 | 3 | 4 |
| 19 | Buffalo milk | Day19 | 6 | 3 | 4 | 3 | 5 | 5 | 3 | 6 | 5 |
| 20 | Buffalo milk | Day20 | 3 | 4 | 5 | 4 | 4 | 4 | 4 | 5 | 3 |
| 21 | Buffalo milk | Day21 | 3 | 6 | 5 | 5 | 4 | 3 | 3 | 4 | 3 |
| 22 | Buffalo milk | Day22 | 3 | 5 | 3 | 5 | 3 | 4 | 4 | 3 | 4 |
| 23 | Buffalo milk | Day23 | 5 | 4 | 3 | 3 | 4 | 3 | 4 | 3 | 4 |
| 24 | Buffalo milk | Day24 | 4 | 3 | 4 | 5 | 4 | 3 | 3 | 3 | 3 |
| 25 | Buffalo milk | Day25 | 4 | 5 | 3 | 4 | 4 | 5 | 5 | 3 | 4 |
| 26 | Buffalo milk | Day26 | 4 | 3 | 6 | 6 | 3 | 6 | 5 | 5 | 4 |
| 27 | Buffalo milk | Day27 | 5 | 3 | 4 | 3 | 4 | 5 | 4 | 4 | 4 |
| 28 | Buffalo milk | Day28 | 6 | 3 | 3 | 4 | 3 | 3 | 4 | 3 | 6 |
| 29 | Buffalo milk | Day29 | 3 | 3 | 5 | 3 | 4 | 4 | 3 | 4 | 3 |
| 30 | Buffalo milk | Day30 | 3 | 4 | 5 | 4 | 3 | 4 | 5 | 4 | 5 |
Table 3: Excretion of Brucella organisms and microbial contaminants identified in milk of buffaloes vaccinated with Brucella abortus S19 reduced dose S/C vaccine.
In the present study, Brucella abortus S19 strain was not isolated from any of the milk samples of vaccinated animals (Table 2 and 3). This is in agreement with the findings of [11] who found all the milk samples negative for Brucella spp. in microbiological culture. Although the milk samples were negative for Brucella abortus organisms, microbial contamination was observed in all the milk samples.Microbial contamination included pathogens like Micrococcus luteus, Pseudomonas fluorescens, Brevundimonas diminuta, Kytococcus sedentarius, Agrobacterium tumefaciens, Streptococcus vestibularis, Sphingomonas paucimobilis, Lactococcus raffinolactis bacteria tend to be released in very large numbers and create elevated counts. The findings were similar to the findings of [12] who found rapid growth of bacterial species under warm conditions to produce high colonies count. The contamination found in the milk samples might generally occur from three main sources; within the udder, exterior to the udder and from the surface of milk handling and storage equipments and the surrounding air, feed, soil, feces and grass [13,14].
It can be concluded for the present study that the Brucella abortus S19 reduced dose subcutaneous vaccine is safe when administered to lactating cattle and buffaloes and will not cause excretion of Brucella organisms in milk.
