The present study is part of an extensive project on infectious cause of reproductive disorders in dairy cattle. During sampling on the present study farm, abortions were encountered and became available for the current investigation.
The farm is located in Mbarali district in Mbeya region in the southern highlands of Tanzania. At the time of sampling the farm had a total of 350 cattle which were crossbreeds of Friesian and Ayrshire with Boran and Zebu, 130 goats, 90 sheep and six dogs. The animals mingled with close interactions among them. All cattle and small ruminants grazed in controlled areas. The study herd had minimal contacts with pastoral herds and other dairy herds around, most of which took place during the dry season. There was no history of vaccination against brucellosis on the study farm.
Samples included serum, milk and one aborted fetus including fetal membranes collected in 2012–2013. Cattle were purposively selected to include only those above 6 months of age, while sheep and goats were randomly selected. Blood samples were collected from 200 cattle aged above 6 months, 50 goats, 35 sheep and six dogs. All female cattle above 6 months of age (n = 187) and all breeding bulls were included (n = 13). About 5 ml of whole blood was collected aseptically into plain vacutainer tubes. Blood samples were left at room temperature for about 12 h to allow serum separation. Serum was then pipetted into sterile tubes. Individual milk samples were collected from 63 cows, altogether from both Rose Bengal Test (RBT) positive and negative cows in sterile containers and properly sealed. Both serum and milk samples were transported to the laboratory on ice and stored at −20 °C until analysis. The aborted fetus and fetal membranes were examined on the farm. The fetus was examined externally for gross lesions and then aseptically dissected for examination of its internal organs. Examination revealed a relatively fresh fetus and its gestation stage was estimated to be 6 months.
Samples from all visceral organs (liver, lungs, kidneys, spleen, heart and brain) including foetal membranes were collected in a sterile plastic bag and were tight sealed and thereafter preserved at −20 °C for bacterial culture and isolation.
The protocol for field studies and collection of animal materials was approved by Njombe and Mbarali districts veterinary and agricultural authorities. Farmers were informed of the study and their verbal consent was sought before commencement of data collection.
Rose Bengal test
All sera from cattle, goats, sheep and dogs were tested for presence of Brucella antibodies using RBT antigen following the manufacturer’s instructions (Standardized B. abortus Rose Bengal Test Antigen Central Veterinary laboratory New Haw Addlestone, Surrey, UK), in accordance with the OIE manual . Brucella positive control serum was always included in the test.
Serum samples from cattle, sheep and goats were analyzed for the presence of Brucella spp. specific antibodies using indirect ELISA commercial kits following manufacturer’s instructions (SVANOVA®
Brucella-Ab I-ELISA Svanova Biotech AB-Uppsala). To monitor interassay variations, Brucella positive control serum was always included.
Milk ring test
Individual milk samples from RBT positive cows were tested on farm using Milk Ring Test (MRT) antigen (Atlas Medical William James House, Cambridge, UK) following the manufacturer’s instructions and in accordance with the OIE manual . Due to shortage of reagents in the field, only ten milk samples were tested.
Bacterial culture, isolation and identification
Bacteriological analysis was performed in a safety level-3 bio-containment facility at the Norwegian Veterinary Institute. Nineteen individual milk samples and aborted fetal organs as well as fetal membranes from one aborted fetus were subjected to bacterial culture. Primary isolation of Brucella spp. was done by inoculating the samples on a Brucella selective media (Selective Serum Dextrose Agar (SSDA)) (Oxoid) and Farrell’s medium. Two plates per sample, (one per medium) were used. From milk samples, 100 μL of milk were inoculated per plate. All plates were incubated both aerobically, and in 5 % CO2 atmosphere, at 37 °C and examined regularly after two, and up to 14 days, for Brucella like colonies. Such colonies were examined further with Gram staining. The plates were discarded if no growth was evident after 14 days of inoculation. Colonies typical of Brucella spp. were sub-cultured from which subsequent bacterial isolates were examined under phase contrast microscope and by Gram staining for organism morphology and size. Typical colonies revealing small Gram-negative coccobacilli, were further analyzed to obtain full identification and biotype.
Classical biotyping was done as described by  at The National Reference Center for Brucellosis, Veterinary and Agrochemical Research Centre (CODA-CERVA) in Belgium. Brucella monospecific antisera A and M and Brucella phages Tb, Wb and Iz obtained from FAO/WHO Collaborating Center for Brucellosis Reference and Research at the Veterinary Laboratory Agency, Weybridge, UK were used. A panel of biotyping tests were performed and interpretation of the results was performed according to the OIE manual .
DNA preparation and PCR
Suspected Brucella spp. isolates were subjected to genomic DNA extraction by heat treating a loopful of bacterial material dissolved in MQ water at 99 °C for 15 min . After centrifugation, the supernatant was used as DNA template.
The extracted DNA was subjected to real time PCR for the Brucella spp. specific targeting IS711 . Primers and probe were developed at the Swedish Institute for Communicable Disease Control (unpublished protocol). Positive results were obtained for the three extracted DNA (results not shown).
Species-level molecular identification was undertaken by multiplex PCR (Bruce-ladder) which was performed as described [23, 24] with the following conditions: Step 1: 95 °C 15 min, Step 2: 94 °C 30 s, Steps 3: 58 °C 90 s, Step 4: 72 °C 3 min, Step 5: 72 °C 10 min. Step 2, 3 and 4 was repeated in 25 cycles. The size of the PCR products was analyzed by capillary electrophoresis with Bioanalyzer®, Agilent Technologies, Santa Clara, CA, USA.
The isolates identified as B. abortus biovar 3 were analyzed using MLVA-VNTR 16 loci as described before . Primers used were those described by Le Fleche et al. . A PCR master mix was prepared using the following reactives: buffer (10×), bethain, dNTP 2.5 mM, Taq DNA polymerase rec (5U/ul Invitrogen), MgCl2 and H2O. The following PCR program with iCycler BioRad was used: Step 1: 96 °C 5 min, Step 2: 96 °C 30 s, Step 3: 60 °C 30 s, Step 4: 70 °C 1 min, Step 5: 70 °C 5 min, Step 6: 8 °C. Step 2, 3 and 4 was repeated in 30 cycles. For the markers bruce 06, bruce 11, bruce 42, bruce 55 with repeat unit size 134 bp, 63 bp, 125 bp and 40 bp respectively, the PCR fragment size was analyzed by 2 % agarose gel electrophoresis. For the markers bruce 08, bruce 12, bruce 43, bruce 45, bruce 18, bruce 19, bruce 21, bruce 04, bruce 07, bruce 09, bruce16 and bruce 30, the size of the PCR products were analyzed by capillary electrophoresis with the CEQ 8000 Genetic Analysis System (Beckman Coulter, Indianapolis, IN, USA). The size of the PCR products were then converted to a corresponding tandem repeat number for each locus as described by Le Fleche et al.  to get the genotype.
To classify the Tanzanian Brucella strains, a polyphasic strategy that included phenotypic (classical biotyping) as well as genomic criteria (presence of IS711, Bruce-ladder and MLVA) was used. Accordingly, MLVA analysis within B. abortus biovar 3 was performed. The profile of the Tanzanian strains were compared to B. abortus biovar 3 genotypes deposited in the Brucella aggregated database on MLVAnet (http://mlva.u-psud.fr/) hosted by the Université Paris-Sud. Four B. abortus biovar 3 genotypes from Belgian strains were also included in the analysis.
Cluster analysis of MLVA data was performed with the software BioNumerics 2.1 (Applied Maths, Sint-Martens-Latem, Belgium) following previous methods by Le Fleche et al. . Cluster analysis was done with Euclidean distance which gives the quantitative difference. Only isolates of 100 % similarity with the same number of tandem repeats in each locus were assigned to the same cluster. The most similar strains clustered closely together with short and thick edges, while the strains with high genomic variations had thin and longer edges. The dendrogram was generated using a distance matrix calculated with the categorical coefficient and the unweighted-pair group method using average linkages as previously described . An identical weight was given to each marker. The MLVA profile of the isolates was also subjected to a minimum spanning tree (MST) analysis in BioNumerics (MLVA plugin 2.1), illustrating the relationship and possible mutation pathways within the clusters based on single locus variations (SLV). Only the units (and not the sizes) from each marker were considered for the analysis. The nodes (circles) consist of identical genotypes and the edges (lines) of weight based on number of mutations (steps) taken from the loci were used. Long weight (steps) indicates multiple mutations while short weight indicates few mutations.