Investigation of foot and mouth disease virus and other animal pathogens in cattle, buffaloes and goats at the interface with Akagera National Park 2017 - 2020.

BACKGROUND: Foot-and-Mouth Disease Virus (FMDV) is a positive-sense RNA virus of the family of the picornaviridæ that is responsible for one of the livestock diseases with the highest economic impact, the Foot-and-Mouth Disease (FMD). FMD is endemic in Rwanda but there are gaps in knowing its seroprevalence and molecular epidemiology. This study reports the FMD seroprevalence and molecular characterization of FMDV in Eastern Rwanda. RESULTS: The overall seroprevalence of FMD in the study area is at 9.36% in cattle and 2.65% in goats. We detected FMDV using molecular diagnostic tools such as RT-PCR and RT-LAMP and the phylogenetic analysis of the obtained sequences revealed the presence of FMDV serotype SAT 2, lineage II. Sequencing of the oropharyngeal fluid samples collected from African buffaloes revealed the presence of Prevotela ruminicola, Spathidium amphoriforme, Moraxella bovoculi Onchocerca flexuosa, Eudiplodinium moggii, Metadinium medium and Verrucomicrobia bacterium among other pathogens but no FMDV was detected in African buffaloes. CONCLUSIONS: We recommend further studies to focus on sampling more African buffaloes since the number sampled was statistically insignificant to conclusively exclude the presence or absence of FMDV in Eastern Rwanda buffaloes. The use of RT-PCR alongside RT-LAMP demonstrates that the latter can be adopted in endemic areas such as Rwanda to fill in the gaps in terms of molecular diagnostics. The identification of lineage II of SAT 2 in Rwanda for the first time shows that the categorised FMDV pools as previously established are not static over time.

Risk factors for the incursion, spread and persistence of the foot and mouth disease virus in Eastern Rwanda.

BACKGROUND: Identification of risk factors is crucial in Foot-and-mouth disease (FMD) control especially in endemic countries. In Rwanda, almost all outbreaks of Foot-and-Mouth Disease Virus (FMDV) have started in Eastern Rwanda. Identifying the risk factors in this area will support government control efforts. This study was carried out to identify and map different risk factors for the incursion, spread and persistence of FMDV in Eastern Rwanda. Questionnaires were administered during farm visits to establish risk factors for FMD outbreaks. Descriptive statistical measures were determined and odds ratios were calculated to determine the effects of risk factors on the occurrence of FMD. Quantum Geographic Information System (QGIS) was used to produce thematic maps on the proportion of putative risk factors for FMD per village. RESULTS: Based on farmers' perceptions, 85.31% (with p < 0.01) experienced more outbreaks during the major dry season, a finding consistent with other reports in other parts of the world. Univariate analysis revealed that mixed farming (OR = 1.501, p = 0.163, CI = 95%), and natural breeding method (OR = 1.626; p = 0.21, CI = 95%) were associated with the occurrence of FMD indicating that the two risk factors could be responsible for FMD outbreaks in the farms. The occurrence of FMD in the farms was found to be significantly associated with lack of vaccination of calves younger than 12 months in herds (OR = 0.707; p = 0.046, CI = 95%). CONCLUSIONS: This is the first study to describe risk factors for persistence of FMDV in livestock systems in Rwanda. However, further studies are required to understand the role of transboundary animal movements and genotypic profiles of circulating FMDV in farming systems in Rwanda.

Molecular prevalence of emerging Anaplasma and Ehrlichia pathogens in apparently healthy dairy cattle in peri-urban Nairobi, Kenya.

BACKGROUND: Anaplasma and Ehrlichia species are tick-borne pathogens of both veterinary and public health importance. The current status of these pathogens, including emerging species such as Ehrlichia minasensis and Anaplasma platys, infecting cattle in Kenya, remain unclear, mainly because of limitation in the diagnostic techniques. Therefore, we investigated the Anaplasma and Ehrlichia species infecting dairy cattle in Nairobi, Kenya using molecular methods. RESULTS: A total of 306 whole blood samples were collected from apparently healthy dairy cattle. Whole blood DNA was extracted and tested for presence of Anaplasma and Ehrlichia DNA through amplification and sequencing of the 16S rDNA gene. Sequence identity was confirmed using BLASTn analysis while phylogenetic reconstruction was performed to determine the genetic relationship between the Kenyan isolates and other annotated genotypes available in GenBank. Anaplasma and Ehrlichia species were detected in 19.9 and 3.3% of all the samples analyzed, respectively. BLASTn analysis of the sequences against non-redundant GenBank nucleotide database revealed infections with A. platys (44.8%), A. marginale (31%) and A. bovis (13.8%). All four sequenced Ehrlichia spp. were similar to Ehrlichia minasensis. Nucleotide polymorphism was observed for A. platys, A. bovis and E. minasensis. The Anaplasma species clustered in four distinct phylogenetic clades including A. marginale, A. platys, A. bovis and some unidentified Anaplasma spp. The Kenyan Ehrlichia minasensis clustered in the same clade with isolates from America and Australia but distant from E. ruminantium. CONCLUSION: This study provides the first report of infection of dairy cattle in Kenya with A. platys and E. minasensis, which are emerging pathogens. We conclude that cattle in peri-urban Nairobi are infected with various species of Anaplasma and E. minasensis. To understand the extent of these infections in other parts of the country, large-scale screening studies as well as vector identification is necessary to inform strategic control.

Detection of Brucella spp. in raw milk from various livestock species raised under pastoral production systems in Isiolo and Marsabit Counties, northern Kenya.

INTRODUCTION: Brucellosis is an important zoonotic disease in Kenya, and identifying the bacteria in milk is important in assessing the risk of exposure in people. METHODS: A cross-sectional study that involved 175 households was implemented in the pastoral counties of Marsabit and Isiolo in Kenya. Pooled milk samples (n = 164) were collected at the household level, and another 372 were collected from domesticated lactating animals (312 goats, 7 sheep, 50 cattle and 3 camels). Real-time polymerase chain reaction (qPCR) testing of the milk samples was performed to identify Brucella species. Brucella anti-LPS IgG antibodies were also detected in bovine milk samples using an indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Based on the qPCR, the prevalence of the pathogen at the animal level (considering samples from individual animals) was 2.4% (95% confidence interval (CI) 1.1-4.5) and 3.0% (CI: 1.0-7.0) in pooled samples. All 14 samples found positive by qPCR were from goats, with 10 contaminated with B. abortus and 4 with B. melitensis. The Brucella spp. antibody prevalence in bovine milk using the milk ELISA was 26.0% (95% CI: 14.6-40.3) in individual animal samples and 46.3% (95% CI: 30.7-62.6) in pooled samples. CONCLUSION: The study is the first in Kenya to test for Brucella spp. directly from milk using qPCR without culturing for the bacteria. It also detected B. abortus in goats, suggesting transmission of brucellosis between cattle and goats. The high prevalence of Brucella spp. is a significant public health risk, and there is a need for intervention strategies necessary in the study area.

Mapping Nairobi's dairy food system: An essential analysis for policy, industry and research.

Demand for dairy products in sub-Saharan Africa, is expected to triple by 2050, while limited increase in supply is predicted. This poses significant food security risk to low income households. Understanding how the dairy food system operates is essential to identify mitigation measures to food insecurity impact. This study aims to determine the structure and functionality of Nairobi's dairy system using a value chain mapping approach. Primary data were gathered through focus group discussions and key informant interviews with dairy value chain stakeholders in Nairobi to obtain qualitative information on people and products in the chains while describing their interactions and flows. Qualitative thematic analysis combined with flowcharts created by participants enabled identification of key food system segments and the development of chain profiles (or flow-diagrams) which together form Nairobi's dairy system. Seven chain profiles forming Nairobi's dairy value chain were identified. These were found to be dominated by small-scale individuals who operate largely independently. Our profiles for the urban and peri-urban farming systems were structurally similar in their downstream networks, obtaining inputs from similar sources. Upstream, the urban systems were shorter, supplying mostly to immediate neighbours or based on own consumption, while the peri urban systems supplied to a wider network and showed some affiliations to producers' associations. Two distinct profiles characterize the milk flow from traders belonging either to a Dairy Traders Association (DTA) or those not belonging to this association (non-DTA). DTA traders sell mainly to fixed retailers and non-DTA traders to mobile retailers (hawkers or roadside vendors). Profiles associated with medium and large cooperatives were driven by networks of collection centres, but with medium-sized cooperatives selling half of their production to large processing companies, and large cooperatives only to fixed retailers. Large processing companies' profiles indicated distribution of high volumes and value addition processing. They reported strategic milk collection arrangements with suppliers on long, medium - or short - term contracts and with well-established product distribution channels. We have identified numerous inter-linkages across dairy chain profiles in Nairobi's complex system, demonstrating significant interdependency among the stakeholders. Therefore, enhancing the system's efficiency requires a holistic, system-wide approach and any policy interventions should consider every segment of the value chain. This study provides a methodological approach for organizations and policy makers to understand and address structural and functional vulnerabilities within food systems more broadly. The insights from this study are relevant to other rapidly growing cities in the region.

Piloting a livestock identification and traceability system in the northern Tanzania-Narok-Nairobi trade route.

We designed and piloted a livestock identification and traceability system (LITS) along the Northern Tanzania-Narok-Nairobi beef value chain. Animals were randomly selected and identified at the primary markets using uniquely coded ear tags. Data on identification, ownership, source (village), and the site of recruitment (primary market) were collected and posted to an online database. Similar data were collected in all the markets where tagged animals passed through until they got to defined slaughterhouses. Meat samples were collected during slaughter and later analyzed for tetracycline and diminazene residues using high-performance liquid chromatography (HPLC). Follow up surveys were done to assess the pilot system. The database captured a total of 4260 records from 741 cattle. Cattle recruited in the primary markets in Narok (n = 1698) either came from farms (43.8%), local markets (37.7%), or from markets in Tanzania (18.5%). Soit Sambu market was the main source of animals entering the market from Tanzania (54%; n = 370). Most tagged cattle (72%, n = 197) were slaughtered at the Ewaso Ng'iro slaughterhouse in Narok. Lesions observed (5%; n = 192) were related to either hydatidosis or fascioliasis. The mean diminazene aceturate residue level was 320.78 ± 193.48 ppb. We used the traceability system to identify sources of animals with observable high drug residue levels in tissues. Based on the findings from this study, we discuss opportunities for LITS-as a tool for surveillance for both animal health and food safety, and outline challenges of its deployment in a local beef value chain-such as limited incentives for uptake.

Identification and characterization of profilin antigen among Babesia species as a common vaccine candidate against babesiosis.

We have characterized a member of the profilin (PROF) family protein as a common antigen in three pathogens-Babesia bovis (B. bovis), Babesia bigemina (B. bigemina), and Babesia microti (B. microti)-and evaluated its immunogenic and cross-protective properties against a challenge infection with B. microti in BALB/c mice. The recombinant PROF proteins of B. bovis, B. bigemina, and B. microti were successfully expressed in Escherichia coli (E. coli) as soluble GST fusion proteins (rBboPROF, rBbigPROF, and rBmPROF, respectively), and they were found to be antigenic. On probing with mouse anti-rPROF serum, green fluorescence was observed on the parasites' cytosols by confocal laser microscopy. Immunization regimes in BALB/c mice using rPROFs induced cross-protective immunity against B. microti infection based on high levels of cytokines and immunoglobulin (IgG) titers, a reduction in peak parasitemia levels, and earlier clearance of the parasite as compared with control mice. The findings of the present study indicate that PROF is a common antigen among bovine and murine Babesia parasites, and it might be used as a common vaccine candidate against babesiosis.

Seroprevalence and risk factors associated with Theileria parva infection among calves in Narok County, Kenya.

Background and Aim: East Coast fever (ECF), caused by Theileria parva, is a devastating disease that causes significant economic losses to cattle production in sub-Saharan Africa. Prevention and control of ECF are challenging in pastoral settings due to inadequate epidemiological information. This study aimed to estimate the seroprevalence and risk factors associated with T. parva infection among calves in different production systems to help design appropriate control interventions. Materials and Methods: Blood samples were collected from 318 calves and tested using an indirect enzyme-linked immunosorbent assay targeting antibodies against polymorphic immunodominant molecules found on the surface of T. parva. Information on calf characteristics and management practices was also collected during sampling. Descriptive statistics and logistic regression were used to analyze potential risk factors, such as age and acaricide application, where p < 0.05 was considered significant. Results: Of the 318 calves sampled, 41 (12.89%) were positive for T. parva, with a higher proportion in pastoral systems (36.58%) than in mixed farming systems (34.10%) and agropastoral systems (29.27%). From univariate analysis, calf age (p = 0.002), body weight (p = 0.001), suckling status (p = 0.026), rectal temperature (p = 0.06), calves on pasture (p = 0.022), other feeds (p = 0.004), feed grown within the farm (p = 0.004), acaricide application (p = 0.001), and acaricide application frequency (p = 0.001) were significantly associated with seropositivity. However, calf age (odds ratio [OR], 0.96; 95% confidence interval [CI], 0.91-0.99; p = 0.04), other feeds (OR, 8.82; 95% CI, 1.74-44.63; p = 0.009), and suckling status (OR, 0.38; 95% CI, 0.15-0.99; p = 0.05) were significantly associated with T. parva infection in the multivariable mixed logistic model. Conclusion: T. parva is circulating in young calves in the study area (and possibly in cattle populations due to maternal transfer of antibodies to the calves). There is a need for molecular surveillance to determine the presence and burden of T. parva infection.

Phenotypic and Genotypic Characterization of Extended Spectrum Beta-Lactamase-Producing Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in Two Kenyan Facilities: A National Referral and a Level Five Hospital.

Background: The emergence of antimicrobial resistance (AMR) and multidrug resistance (MDR) among Escherichia coli and Klebsiella pneumoniae, especially through the production of extended spectrum ß-lactamases (ESBLs), limits therapeutic options and poses a significant public health threat. Objective: The aim of this study was to assess the phenotypic and genetic determinants of antimicrobial resistance of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates from patient samples in two Kenyan Hospitals. Methods: We collected 138 E. coli and 127 K. pneumoniae isolates from various clinical specimens at the two health facilities from January 2020 to February 2021. The isolates' ESBL production and antibiotic susceptibility were phenotypically confirmed using a standard procedure. Molecular analysis was done through conventional polymerase chain reaction (PCR) with appropriate primers for gadA, rpoB, blaTEM, blaSHV, blaOXA, blaCTX-M-group-1, blaCTX-M-group-2, blaCTX-M-group-9, and blaCTX-M-group-8/25 genes, sequencing and BLASTn analysis. Results: Most E. coli (82.6%) and K. pneumoniae (92.9%) isolates were ESBL producers, with the highest resistance was against ceftriaxone (69.6% among E. coli and 91.3% among K. pneumoniae) and amoxicillin/clavulanic acid (70.9% among K. pneumoniae). The frequency of MDR was 39.9% among E. coli and 13.4% among K. pneumoniae isolates. The commonest MDR phenotypes among the E. coli isolates were CRO-FEP-AZM-LVX and CRO-AZM-LVX, while the FOX-CRO-AMC-MI-TGC-FM, FOX-CRO-FEP-AMC-TZP-AZM-LVX-MI and CRO-AMC-TZP-AZM-MI were the most frequent among K. pneumoniae isolates. Notably, the FOX-CRO-FEP-AMC-TZP-AZM-LVX-MI phenotype was observed in ESBL-positive and ESBL-negative K. pneumoniae isolates. The most frequent ESBL genes were blaTEM (42%), blaSHV (40.6%), and blaOXA (36.2%) among E. coli, and blaTEM (89%), blaSHV (82.7%), blaOXA (76.4%), and blaCTX-M-group-1 (72.5%) were most frequent ESBL genes among K. pneumoniae isolates. The blaSHV and blaOXA and blaTEM genotypes were predominantly associated with FOX-CRO-FEP-MEM and CRO-FEP multidrug resistance (MDR) and CRO antimicrobial resistance (AMR) phenotypes, among E. coli isolates from Embu Level V (16.7%) and Kenyatta National Hospital (7.0%), respectively. Conclusions: The high proportion of ESBL-producing E. coli and K. pneumoniae isolates increases the utilization of last-resort antibiotics, jeopardizing antimicrobial chemotherapy. Furthermore, the antimicrobial resistance patterns exhibited towards extended-spectrum cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, fluoroquinolones, and macrolides show the risk of co-resistance associated with ESBL-producing isolates responsible for MDR. Hence, there is a need for regular surveillance and implementation of infection prevention and control strategies and antimicrobial stewardship programs.

Identification and characterization of an interspersed repeat antigen of Babesia microti (BmIRA).

In this report, a novel gene encoding an interspersed repeat antigen from Babesia microti (BmIRA) was identified and described. The full-length cDNA containing an open reading frame of 1,947 bp was obtained by immunoscreening a B. microti cDNA expression library. The full-length of BmIRA gene was expressed as a GST fusion recombinant BmIRA (rBmIRA) in Escherichia coli. Sera of mice immunized with the rBmIRA detected a native parasite protein with a molecular mass of 76 kDa on Western blot analysis. The same protein was detected in the parasites by immunofluorescent antibody test (IFAT). An enzyme-linked immunosorbent assay (ELISA) using rBmIRA detected specific antibodies as early as 11 days post-infection in sera from a hamster experimentally infected with B. microti Gray stain (US type). Furthermore, a rapid immunochromatographic test (ICT) using rBmIRA detected specific antibodies in a hamster experimentally infected with B. microti from day 11 to at least day 180 post-infection. The results indicate the antibody response against the rBmIRA was maintained during the chronic stage of infection. On the other hand, an immunoprotective property of rBmIRA as a subunit vaccine was evaluated in hamsters against B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmIRA could be a useful serodiagnostic antigen for screening of B. microti infection.

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.

Antimicrobial Usage, Susceptibility Profiles, and Resistance Genes in Campylobacter Isolated from Cattle, Chicken, and Water Samples in Kajiado County, Kenya.

Campylobacter organisms are the major cause of bacterial gastroenteritis and diarrhoeal illness in man and livestock. Campylobacter is growingly becoming resistant to critically crucial antibiotics; thereby presenting public health challenge. This study aimed at establishing antimicrobial use, susceptibility profiles, and resistance genes in Campylobacter isolates recovered from chicken, cattle, and cattle-trough water samples. The study was conducted between October 2020 and May 2022 and involved the revival of cryopreserved Campylobacter isolates confirmed by PCR from a previous prevalence study in Kajiado County, Kenya. Data on antimicrobial use and animal health-seeking behaviour among livestock owners (from the same farms where sampling was done for the prevalence study) were collected through interview using a pretested semistructured questionnaire. One hundred and three isolates (29 C. coli (16 cattle isolates, 9 chicken isolates, and 4 water isolates) and 74 C. jejuni (38 cattle isolates, 30 chicken isolates, and 6 water isolates)) were assayed for phenotypic antibiotic susceptibility profile using the Kirby-Bauer disk diffusion method for ampicillin (AX), tetracycline (TE), gentamicin (GEN), erythromycin (E), ciprofloxacin (CIP), and nalidixic acid (NA). Furthermore, detection of genes conferring resistance to tetracyclines (tet (O), ß-lactams (bla OXA-61), aminoglycosides (aph-3-1), (fluoro)quinolones (gyrA), and multidrug efflux pump (cmeB) encoding resistance to multiple antibiotics was detected by mPCR and confirmed by DNA sequencing. The correlation between antibiotic use and resistance phenotypes was determined using the Pearson's correlation coefficient (r) method. Tetracyclines, aminoglycosides, and ß-lactam-based antibiotics were the most commonly used antimicrobials; with most farms generally reported using antimicrobials in chicken production systems than in cattle. The highest resistance amongst isolates was recorded in ampicillin (100%), followed by tetracycline (97.1%), erythromycin (75.7%), and ciprofloxacin (63.1%). Multidrug resistance (MDR) profile was observed in 99 of 103 (96.1%) isolates; with all the Campylobacter coli isolates displaying MDR. All chicken isolates (39/39, 100%) exhibited multidrug resistance. The AX-TE-E-CIP was the most common MDR pattern at 29.1%. The antibiotic resistance genes were detected as follows: tet (O), gyrA, cmeB, bla OXA-61 , and aph-3-1 genes were detected at 93.2%, 61.2%, 54.4%, 36.9%, and 22.3% of all Campylobacter isolates, respectively. The highest correlations were found between tet (O) and tetracycline-resistant phenotypes for C. coli (96.4%) and C. jejuni (95.8%). A moderate level of concordance was observed between the Kirby-Bauer disk diffusion method (phenotypic assay) and PCR (genotypic assay) for tetracycline in both C. coli (kappa coefficient = 0.65) and C. jejuni (kappa coefficient = 0.55). The study discloses relatively high resistance profiles and multidrug resistance to antibiotics of critical importance in humans. The evolution of the multidrug-resistantCampylobacter isolates has been linked to the use and misuse of antimicrobials. This poses a potential hazard to public and animal health, necessitating need to reduce the use of antibiotics in livestock husbandry practice coupled with stringent biosecurity measures to mitigate antimicrobial resistance.

Molecular Prevalence and Risk Factors of Campylobacter Infection in Puppies in the Nairobi Metropolitan Region, Kenya.

Campylobacter species are widely distributed pathogens; however, data on its epidemiology in puppies remain scanty, especially in Kenya. A cross-sectional study was conducted in the Nairobi Metropolitan Region to determine molecular prevalence and associated risk factors of Campylobacter species infection in puppies. A total of 260 rectal swabs were collected from puppies from breeding kennels, shelters, and the University of Nairobi Veterinary Teaching and Referral Hospital. The samples were subjected to polymerase chain reaction (PCR) assays for identification of Campylobacter species. Data on potential risk factors associated with puppy exposure were collected using a semistructured questionnaire. Multivariable mixed effects logistic regression analyses were performed with kennels as random effects. Campylobacter species were detected in 64 of the 260 sampled puppies yielding an overall prevalence of 24.6%. Multivariable results showed that puppies from shelters, puppies from kennels that are washed daily, puppies with a recent history of vomiting, and those treated with antibiotics in the past month were significantly associated with the presence of Campylobacter species. Being a kenneled puppy and having had concurrent bacterial infections were identified as protective factors. This study provides molecular evidence of puppy exposure to Campylobacter species which could have impact on puppy health and highlights the need to develop awareness and management strategies to potentially reduce the risk of transmitting this pathogen among puppies, to humans, and other animals.

Seroprevalence and spatial distribution of livestock brucellosis using three serological tests in Kajiado County, Kenya.

Background: Brucellosis is a serious zoonotic infection with a global socioeconomic impact on both the livestock industry and human health. In Kenya, brucellosis is endemic but there is limited information on the true burden of the disease due to weak or peace-meal surveillance. The true burden and spread of animal brucellosis in Kajiado County is not known. Aim: The aim of the study was to determine the current seroprevalence and spatial distribution of livestock brucellosis in Kajiado County and also to compare the three serological tests, namely; Rose Bengal plate test (RBPT), indirect enzyme-linked immunosorbent assay (i-ELISA), and competitive ELISA (c-ELISA) in the detection of seropositive animals. Methods: A cross-sectional study was undertaken in 5 sub-counties and 13 wards, where a total of 782 serum samples from unvaccinated bovine (n = 278; 34 herds), ovine (n = 256; 25 flocks), and caprine (n = 248; 28 flocks), were screened for anti-Brucella antibodies using RBPT, i-ELISA, and c-ELISA tests, in parallel. Results: Overall animal seroprevalence was 6.91% (54/782); while that for bovine, ovine, and caprine was 18.35% (51/278), 0.78% (2/256), and 0.4% (1/248), respectively. Bovine seroprevalence was 2.2% (6/278), 14.4% (40/278), and 4.7% (13/278) in RBPT, i-ELISA, and c-ELISA tests, respectively; while ovine 0.78% (2/256) and caprine 0.4% (1/248) were positive only in c-ELISA. Bovine herd seropositivity was 67.65% (23/34), whereas ovine and caprine flock seropositivities were 8% (2/25) and 3.6% (1/28), respectively. Conclusion: The findings indicate a moderate seroprevalence of brucellosis in bovine, while that of ovine and caprine was low in Kajiado County. Indirect ELISA was found superior to both c-ELISA and RBPT in detecting bovine seropositive animals, while c-ELISA was superior to both RBPT and i-ELISA in detecting seropositive ovines and caprines. These results will contribute to baseline data for further study of Brucella infection and a starting point for the formulation of a strategy for the control of brucellosis in Kajiado County.

Macrophages are critical for cross-protective immunity conferred by Babesia microti against Babesia rodhaini infection in mice.

Although primary infection of mice with Babesia microti has been shown to protect mice against subsequent lethal infection by Babesia rodhaini, the mechanism behind the cross-protection is unknown. To unravel this mechanism, we investigated the influence of primary infection of mice with nonlethal B. microti using different time courses on the outcome of subsequent lethal B. rodhaini infection. Simultaneous infections of mice with these parasites resulted in rapid increases in parasitemia, with 100% mortality in BALB/c mice, as observed with control mice infected with B. rodhaini alone. In contrast, mice with acute, resolving, and chronic-phase B. microti infections were completely protected against B. rodhaini, resulting in low parasitemia and no mortalities. Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses were induced. Interestingly, the protected mice had significantly decreased levels of antibody response, cytokines (including gamma interferon [IFN-γ], interleukin-2 [IL-2], IL-8, IL-10, and IL-12), and nitric oxide levels after infection with B. rodhaini. SCID mice and IFN-γ-deficient mice with chronic B. microti infections demonstrated protective responses comparable to those of immunocompetent mice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely, macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and cytokines profiles, indicating that macrophages contribute to the protection against this challenge infection. We conclude that future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of macrophages.

A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model.

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.

Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand.

Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.

Molecular Detection and Characterization of Pasteurella multocida Infecting Camels in Marsabit and Turkana Counties, Kenya.

Pasteurella multocida infection is common in Kenya though there is little knowledge of the genetic diversity of the pathogen. P. multocida is part of the normal flora in the respiratory tract of camels, but it becomes pathogenic when the resistance of the camel body is diminished by bad ecological conditions. This study was conducted to detect, characterize, and determine the genetic diversity of P. multocida infecting camels in Marsabit and Turkana Counties. The KMT1 gene was targeted as the marker gene for P. multocida and hyaD-hyaC, bcbD, dcbF, ecbJ, and fcbD as marker genes for capsular serogroups A, B, D, E, and F, respectively. Out of 102 blood and 30 nasal swab samples, twenty-one samples (16%) were confirmed to be positive for P. multocida and only capsular group E was detected in both counties. The P. multocida sequences were highly conserved and were related to strains from other parts of the world. Our study has confirmed that camels in Marsabit and Turkana Counties of Kenya are infected by P. multocida of capsular type E. Farmers should not underfeed camels, ensure appropriate medication and vaccination programs, and minimize herding of camels in crowded areas especially in wet conditions in order to slow the spread of P. multocida infection.

Beta hemolysin gene of Staphylococcus phage 3AJ_2017 genome is a suitable molecular marker for identification and characterization of pathogenic Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus cause diseases both in humans and animals. These diseases range from mild to fatal infections thus necessitating development of a specific molecular method for detection of pathogenic S. aureus. OBJECTIVES: To identify and analyze genetic profile of pathogenic S. aureus using bacteriophage based genetic biomarkers. METHODS: Using culture and biochemical methods, 148 S. aureus (87 %) were isolated from 170 raw milk samples taken from 10 dairy farms in Marsabit and Isiolo counties in Northern Kenya between June 2016 and February 2017. The samples were collected directly from dairy lactating cows previously diagnosed with S. aureus in a follow-up study. The isolates were analyzed by PCR and sequencing of beta hemolysin (hlb) gene. The genetic relationship between five Kenyan S. aureus isolates and five isolates previously identified was inferred. RESULTS: From the 96 isolates screened for hlb gene, 75 (78.1%) tested positive. Some of the positive isolates yielded a band size of 975 bp, while others 1100 bp. Through Basic Local Alignment Search Tool (BLAST) search analysis, the two different band sizes (975 bp and 1100 bp) were both confirmed to be hlb gene from S. aureus isolates indicating that the difference in band size may have been due to deletions that were detected in the 975 bp hlb gene. Some S. aureus isolates from Kenya appeared to be closely related to isolates from other parts of the world, while some showed a distant relationship. CONCLUSIONS: Phage-derived hlb gene is a suitable molecular marker for detection of pathogenic S. aureus.

Seasonal Prevalence and Molecular Identification of Thermophilic Campylobacter from Chicken, Cattle, and Respective Drinking Water in Kajiado County, Kenya.

Thermophilic Campylobacter species are a leading cause of human gastroenteritis throughout the world and have been implicated in reproductive disorders (abortion), mastitis, enteritis, and/or diarrhoea in livestock. A cross-sectional survey was conducted in Kajiado County to determine prevalence, seasonality, and molecular detection of thermophilic Campylobacter species (with emphasis on C. jejuni, C. coli, and other thermophilic Campylobacter species) in chicken, cattle, and respective pooled drinking water. A total of 457 samples comprising 265 cattle rectal swabs, 142 chicken cloacal swabs, and 50 trough water samples were collected from 55 randomly selected smallholder farms. Individual samples were subjected to standard techniques for isolation and biochemical tests, followed by singleplex polymerase chain reaction (sPCR) assays for identification and confirmation of genus and species. Overall, thermophilic Campylobacter prevalence was 35.4% (95% confidence interval (95% CI) = 31.0-39.8), with C. jejuni dominating at 55.6% (95% CI = 47.9-63.3%) over C. coli in all sample types. The highest thermophilic Campylobacter prevalence was observed in cloacal swabs of live chicken at 44.4% (95% CI = 36.2-52.6%), followed by rectal swabs from live cattle at 30.9% (95% CI = 25.3-36.5%). Water samples from cattle drinkers/trough were found to be contaminated at 34% (95% CI = 20.9-47.1%). The isolation rate was higher in cattle under the confinement system (44.3%) (95% CI = 36.1-52.5%) than in those under the free-roaming grazing system. Thermophilic Campylobacter species were isolated in both seasons, with higher prevalence (39.8% (95% CI = 33.6-45.9)) recorded during rainy and cold season in all sample types except for water. There was significant (P < 0.05) association between season and thermophilic Campylobacter occurrence, even though there were no statistical differences in the prevalence values across the two seasons. Results of this study demonstrate that cattle, chicken, and respective drinking water harbour potentially pathogenic thermophilic campylobacters, with C. jejuni being widely distributed among farms. It is possible that seasonal variations and cattle confinement result in differences in thermophilic Campylobacter carriage. Further epidemiological and phylogenetic studies comparing distribution of thermophilic Campylobacter spp. isolates in livestock, environmental, and human samples are recommended to establish source attribution to reduce the impact of resultant diseases for the wellbeing of public and livestock.