Serological, Cultural, and Molecular Evidence of Brucella Infection in Small Ruminants in Pakistan

Introduction: The objectives of the present study were to determine the seroprevalence and identify the causative agent of brucellosis in small ruminants in Pakistan. Methodology: A total of 278 serum and 212 milk samples were collected from sheep and goats that had close contact with seropositive bovine herds. Data related to age, sex, location, and breed were collected on the sampling day. Serum and milk samples were initially screened using two different Rose Bengal plate test (RBPT) antigens and a milk ring test (MRT). Seropositive samples were subjected to bacterial isolation and PCR analysis using Brucella genus-specific (bcsp31) and Brucella species-specific (IS711 for Brucella abortus and Brucella melitensis) quantitative real-time polymerase chain reactions (qRT-PCR). Results: Twenty-four (8.6%) serum samples were positive by RBPT. Twenty (9.4%) animals were positive for Brucella antibodies using MRT. No Brucella isolates were obtained from the examined blood and milk samples. Of the 24 seropositive serum samples, 18 (75%) were positive in the Brucella genus-specific (bcsp31) and Brucella abortus-specific (IS711) qRT-PCR, respectively. Conclusions: Brucella abortus was identified as causative agent of ovine and caprine brucellosis in Pakistan. Results of this study can be used for the development of an effective control and eradication strategy for brucellosis in livestock, especially small ruminants.


Introduction
Brucellosis is an important zoonotic disease that affects a wide range of animal species as well as humans [1,2].The causative agent of brucellosis in goats and sheep is Brucella melitensis, while B. abortus, B. ovis, and B. suis may cause infections only under certain condition [3][4][5].The major clinical manifestations of brucellosis in sheep and goats are abortion during the last two months of pregnancy, arthritis, fetal membrane retention, weak offspring, orchitis, epididymitis, and sterility [6].
In Pakistan's neighboring countries (Afghanistan, China, India, and Iran), there is evidence of brucellosis in animals and humans [7].Pakistan is an agriculturebased country, and livestock plays a crucial role in people's livelihood, especially people living in rural areas.Among the different livestock species in Pakistan are 28.1 million sheep and 61.5 million goats [8].B. abortus biotype 1 is known to be the causative agent of brucellosis in cattle and buffaloes [2].Quantitative real-time polymerase chain reaction (qRT-PCR) assays have confirmed B. abortus as the causative agent of human brucellosis in high-risk occupations in Pakistan [1].However, limited information based only on serological studies is available on brucellosis in small ruminants (i.e., sheep and goats) in Pakistan.In the Punjab region of Pakistan, 31 (1.46%) sheep and 29 (1.93%) goats were positive for Brucella antibodies [9].
In order to develop effective control and eradication programs, it is essential to establish the causative agent of ovine and caprine brucellosis in Pakistan.Polymerase chain reaction is an appropriate and rapid method for the correct diagnosis of brucellosis instead of the tedious cultivation of the agent [10,11].Due to the economic importance of brucellosis, the objectives of the present study were to determine the seroprevalence and isolate and identify the agent using qRT-PCR assays.

Origin of animals and sampling sites
The study area (the Potohar Plateau, including Rawat, Islamabad, and Kherimurat) was chosen because Brucella abortus was recently identified there to be the causative agent of bovine and human brucellosis.Sheep and goats that had close contact with seropositive bovine herds (i.e., shared the same housing and grazing area) were selected for sampling.Twelve sheep and goat herds (four from Rawat, two from Islamabad, and six from Kherimurat) were selected.Herds with sheep (n = 2), goats (n = 3), and with both sheep and goats (n = 7) were investigated.Samples were collected from every adult animal in each herd.Herds in Rawat and Kherimurat were large (> 20 animals).A total of 278 (66 male and 212 female) animals were selected for blood collection (118 sheep and 160 goats).
Milk samples (n = 212) were collected only from females that were already selected for blood sampling.On the sampling day, data related to farm location, sex, species, and breed were also collected.

Blood and milk collection and serology
Milk and blood samples were collected, stored, and processed according to standard procedures [12].Milk samples were collected from both quarters.Serum samples were initially screened with two Rose Bengal plate test (RBPT) antigens: the antigen of the Veterinary Research Institute (VRI), Lahore, Pakistan, and the antigen of IDEXX, Pourquier, France.Briefly, 30 µL of serum was mixed with an equal volume of antigen preparation on a glass plate; the plate was agitated gently for 4 minutes.A serum sample was considered positive if agglutination occurred.The milk ring test (MRT) antigen used was purchased by VRI Lahore, Pakistan.In brief, MRT antigen was brought to room temperature before use.One milliliter of milk was added to a test tube.Then, 30 to 40 µL of antigen was added, and the sample was mixed and incubated at 37°C for 1 hour.A sample that had blue colouration at the surface was considered to be positive.Then, serum samples were shipped on dry ice to the Friedrich-Loeffler-Institute (FLI), Germany, for molecular analysis.

Bacteriology
Culturing of brucellae from seropositive blood (n = 24) and milk samples (n = 20) was done at the bacteriology section, National Veterinary Laboratory, Islamabad, Pakistan, on modified Farrell's serum dextrose agar according to standard procedures [12,13].Modified Farrell's serum dextrose agar with 5% horse serum, 1% dextrose, and the following antibiotics (added to 1 L medium): cycloheximide (100 mg), bacitracin (25,000 IU), polymyxin B sulfate (5,000 IU), vancomycin (20 mg), nalidixic acid (5 mg), and nystatin (100,000 IU), was used for primary isolation of brucellae.Plates were inoculated with blood and milk and incubated aerobically and in the presence of 5%-10% carbon dioxide at 37°C.The plates were checked for up to 10 days for the presence of bacterial growth.

DNA extraction from serum samples
DNA was extracted from all seropositive serum samples using high Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions.Purity and concentration of DNA was tested using a Nano-Drop ND-1000 UV-Vis spectrophotometer (Nano-

Molecular detection by qRT-PCR
All DNA samples were analyzed by Brucella genus-specific (bcsp31) and species-specific real-time PCR assays for B. abortus and B. melitensis [14].The detailed procedure used has been described previously [11].The details of primers and probes are given in Tables 1 and 2 [14].Amplification was done in 25 µL of total volume.Nuclease-free water, DNA of E. coli (DSM 30083, ATCC 11775), and DNA of Brucella reference strains (BA 544, BM 16M) were used as negative control (NC), no template positive control (NPC), and positive control, respectively.The amplification reactions were done in duplicate in optical 96-well microplates (Thermo Fisher Scientific Inc., Waltham, USA) using a Mx3000P thermocycler system (Agilent Technologies, Santa Clara, USA).Cycling conditions were as follows: one cycle for decontamination at 50°C for 2 minutes, initial denaturation at 95°C for 10 minutes, followed by 50 cycles at 95°C for denaturation for 25 seconds, and 1 minute for annealing at 57°C.No internal

Results
A total of 24 (8.6%) and 21 (7.6%) serum samples were found to be positive for Brucella antibodies using IDEXX and VRI RBPT antigens, respectively.Twenty (9.4%) animals were positive for Brucella antibodies using MRT (Table 3).Based on parallel interpretation of RBPT antigens, female animals (10.4%) were found to be more often seropositive than were males (3.03%).Moreover, 7 (58.3%) of 12 herds were positive for Brucella infection.
Animals kept in Rawat region were more often seropositive than animals kept in Islamabad and Kherimurat.The seroprevalence of Brucella antibodies was higher in goats than in sheep.Of the two breeds of sheep investigated, only 3 (3.6%)Salt Range were found to be seropositive.In goats, the prevalence was slightly higher in Beetal and local hairy goats.No isolate of Brucella was recovered from blood and milk samples of sheep and goats.Of the 24 seropositive samples, 18 (75%) were positive in the Brucella genus-specific (bcsp31) qRT-PCR.The serum samples positive with the Brucella genus-specific (bcsp31) qRT-PCR were also positive with the Brucella abortus-specific (IS711) qRT-PCR (Table 4).None of the serum samples was positive for Brucella melitensis DNA.Both male and female animals were found positive for brucellosis in qRT-PCR.

Discussion
Brucellosis is an infectious disease found in a wide range of animal species; is also transmitted to humans via secretions and excretions of infected animals.For the development and onset of control and eradication programs, knowledge of the prevalent species and biotypes is important in order to understand the chains of infection.This study provides first evidence that Brucella abortus is the causative agent of brucellosis in small ruminants in Pakistan.
A total of 24 (8.6%) serum samples were found to be positive for Brucella antibodies in the present study.These data are in contrast to a previous study conducted in the Punjab region of Pakistan, when a seroprevalence of only 1.6% was reported in small ruminants [9].A comparable high prevalence (11.2%) was found in livestock research stations of the Punjab region of Pakistan [15].
A reason for the higher seropositivity of animals in this study is that these small ruminants had close contact with seropositive herds of cattle and buffalo, a possible source of Brucella spp.It is also common practice that livestock farmers do not cull and dispose of brucellosis-positive animals, but rather sell them to other farmers, thereby enabling infected animals to enter Brucella-free herds and transmit infection to other animals.Thus, prevalence of brucellosis is increasing day by day in Pakistan.
In this study, the seroprevalence was higher in goats than in sheep.These results are in line with previous studies in Pakistan [9,15].In previous studies conducted in various countries (Bangladesh, Kosovo, Sudan, Nigeria), the same trend was noticed [16][17][18][19].Higher seroprevalence in sheep was only reported from Egypt and Tajikistan [20,21].This variation might be due to the differences in countries' local herd management systems.
The seroprevalence was higher in female animals than in male animals.A higher seroprevalence in female sheep and goats has been reported from the district of Peshawar, Pakistan [22], whereas higher seropositivity in male sheep and goats has been reported in goats in Bangladesh and Mexico [23,24].In the present study, the number of male animals was lower than the number of female animals because female animals were served by positive rams of the same herd or were of older age, increasing the risk of getting infected.
The seroprevalence in sheep and goats of the three regions investigated varied greatly.The highest seroprevalence was reported from Rawat (9.8%), followed by Kherimurat (9.4%) and Islamabad (4%).One of the possible reasons for the high prevalence in Rawat and Kherimurat is that serum samples were collected from large herds.It is a well-known fact that larger herds have a higher probability of contacting infected animals.Herd prevalence is associated considerably with herd size [25].Variation in seropositivity was also seen in goats in Mexico [23].
In the present study, two breeds of goats (Beetal and local hairy) were investigated; the prevalence was different in both breeds.Differences in goat breeds were also reported from Mexico [23].However, it was surprising to find that only one breed of sheep was infected (Salt Range).Brucellosis-negative sheep of the Afghani breed also shared the same grazing area and housing with seropositive Salt Range sheep.The most likely reason for this finding is that all of these animals were males and were reared to be sold to farmers for breeding.
No isolates of Brucella were recovered from milk and blood samples of sheep and goats in the present study using Farrell's modified serum dextrose agar.This agar has been used successfully to isolate Brucella abortus biotype 1 from milk samples of sheep from Nigeria [26,27].The possible reason for failure of culture is that the animals were infected chronically and so no living bacteria were circulating any longer.
Molecular detection of Brucella genus DNA based on qRT-PCR (bcsp31) from serum samples confirmed the presence of Brucella in these sheep and goat serum samples.Bcsp31 qRT-PCR and other PCR assays have been used by various authors in different countries to amplify Brucella DNA from blood, milk, and serum [28][29][30], and PCR is a validated technique in the of brucellosis.Although species-specific PCR assays have a lower analytical sensitivity than do genus-specific PCRs, B. abortus was identified as the causative agent of brucellosis in sheep and goats.Interestingly, no evidence of B. melitensis infection was found.B. abortus was confirmed as the causative agent of ovine and caprine brucellosis in previous studies using PCR assays [31] and of sheep brucellosis using bacteriology [32][33][34].

Conclusions
B. abortus has recently been identified as the causative agent of bovine and human brucellosis in the Potohar Plateau of Pakistan.No evidence for B. melitensis was found in this study.A possible reason for this is that the present study focused on small ruminants that had close contact with cattle and buffalo herds infected with B. abortus.Moreover, only a small number of samples was collected for this study.In future studies, after involving large numbers of samples and new study areas, B. melitensis may also be added to the list of prevalent Brucella species in small ruminants of Pakistan.We have demonstrated that there is an immense need to develop a control and eradication program that includes vaccination, screening, and culling of brucellosis-positive cattle, sheep, and goats.Further studies will also have to include camels.

Table 1 .
Primers for Brucella genus and species-specific qRT-PCR

Table 2 .
Probes for Brucella genus and species-specific qRT-PCR

Table 3 .
Comparative results of serological tests for samples of sheep and goat sera and milk RBPT: rose Bengal plate test; MRT: milk ring test; VRI: veterinary research institute, Pakistan

Table 4 .
Comparative results of bacteriology and molecular methods for samples of sheep and goats