TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples.

Introduction: Canine leptospirosis has always been a differential diagnosis in dogs presenting with clinical signs and blood profiles associated with kidney and/or liver disease. The conventional polymerase chain reaction (PCR) provides diagnoses, but real-time PCR-based tests provide earlier confirmation and determine the severity of infection, especially in the acute stage, allowing early detection for immediate treatment decisions. To our knowledge, real-time PCR has not been routinely adopted for clinical investigation in Malaysia. This study evaluated TaqMan real-time PCR (qPCR) assays diagnosing leptospirosis and compared their applicability to clinical samples from dogs with kidney and/or liver disease against a conventional PCR reference. Material and Methods: The qPCR assays were validated using existing leptospiral isolates. Whole blood and urine samples were analysed using a conventional PCR, LipL32(1) and LipL32(2) qPCRs and a microscopic agglutination test. The sensitivity and specificity of the qPCRs were determined. Results: The LipL32(1) qPCR assay had more diagnostic value than the LipL32(2) qPCR assay. Further evaluation of this assay revealed that it could detect as low as five DNA copies per reaction with high specificity for the tested leptospiral strains. No cross-amplification was observed with other organisms. Analysing the clinical samples, the LipL32(1) qPCR assay had 100.0% sensitivity and >75.0% specificity. Conclusion: The LipL32(1) qPCR assay is sensitive, specific and has the potential to be applied in future studies.

Risk and Predictive Factors of Leptospirosis in Dogs Diagnosed with Kidney and/or Liver Disease in Selangor, Malaysia.

Canine leptospirosis is commonly associated with kidney and/or liver disease. It has been widely reported and causes public health concerns due to its zoonotic potential and its re-emergence, resulting from close contact between humans and dogs. The current study identified potential risk and predictive factors for dogs diagnosed with kidney and/or liver disease due to leptospirosis. A total of 124 client-owned dogs were recruited, and information such as signalment, medical history, management, and clinical findings were documented. Samples collected from the recruited dogs were directly tested using polymerase chain reaction (PCR) and subsequently inoculated for bacterial isolation. Statistical analyses were descriptively analyzed, and risk analyses were performed using Pearson chi-square tests and logistic regression. A total of 53 dogs (42.7%) were positive for leptospiral infection based on PCR, and 10 leptospiral isolates were successfully recovered from eight dogs. The mortality rate of infected dogs was 34.0% (18/53). Medium and large dog breeds, with a history of exposure to rats, and managed outdoors had a greater risk for leptospirosis (p < 0.05). The significant predictors for the dogs' positivity were the presence of rats and acute clinical illness (p < 0.05). Administration of antibiotics and the detection of clinical illness at an early stage of the disease improved the survivability of the dogs (p < 0.05). Identifying the profile of dogs that are at risk to leptospirosis could be useful in the design of diagnostic and treatment strategies, as well as to increase awareness for prevention of the disease.

Shedding and Genetic Diversity of Leptospira spp. From Urban Stray Dogs in Klang Valley, Malaysia.

Leptospirosis is an endemic zoonoses of global proportions. Stray dogs have been postulated to play a role in disease transmission; however, supporting information are still limited. Roaming behavior may not only predispose the dogs to infection, but could also contribute to disease spread. In this study, the susceptibility of urban stray dogs in shedding Leptospira spp. was determined. Blood, urine, and tissue samples of kidney and liver were collected from 100 dogs from 2 animal control facilities. Serological testing using microscopic agglutination test (MAT) were performed on blood against 20 leptospiral serovars with a cut-off titre of ≥ 1:100. Samples were cultured onto semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media. Isolates were identified using molecular polymerase chain reaction (PCR) using 2 primers (16s rRNA and LipL32) and hyperimmune serum (HIS) MAT. The seroprevalence for the dogs positive for leptospirosis was 32% (n=32/100) with the following detected serovars: Javanica (n=13), Bataviae (n=10), Icterohaemorrhagiae (n=3), Autumnalis (n=2), Canicola (n=1), Pyrogenes (n=1), Copenhageni (n=1), and Australis (n=1). Six Leptospira spp. isolated were procured from urine (n=2), kidney (n=2) and liver (n=2). All 6 isolates belonged to L. interrogans, a pathogenic variant of Leptospira spp. Serotyping and phylogenetic analysis suggested serovar Bataviae (n=5) and serovar Canicola (n=1). Presence of vaccinal serovars (Icterohaemorrhagiae and Canicola) suggested potential post-vaccination antibodies but the predominance of non-vaccinal serovars (Javanica and Bataviae) indicate the possibility of current infection or post-exposure. Isolation of Leptospira spp. directly from urine sample not only suggested an active infection but highlighted the potential shedding capability among these stray dogs. These findings further strengthen speculations that urban stray dogs could play a role in transmission and dissemination of leptospirosis through their constant movement. The urine of infected dogs may contaminate the environment, posing a major public health threat.

Detection and characterization of Leptospira spp. in dogs diagnosed with kidney and/or liver disease in Selangor, Malaysia.

Leptospirosis is a serious bacterial disease that affects both humans and animals. A wide range of symptoms have been described in humans; the disease in dogs is commonly associated with kidney and/or liver disease. In Malaysia, information about the common serovars infecting dogs is limited. Therefore, we investigated the occurrences of leptospirosis in 124 pet dogs diagnosed with kidney and/or liver disease. Blood, urine, abdominal effusion, and/or kidney and liver were collected from the dogs. Based on microscopic agglutination testing, 53 of 124 (42.7%) dogs were seropositive for leptospiral exposure. Sera were frequently positive to serovars Bataviae (n = 12), Javanica (n = 10), and Icterohaemorrhagiae (n = 10). Direct detection using PCR showed that 42 of 124 (33.9%) of the whole blood and 36 of 113 (31.9%) urine samples were positive for pathogenic Leptospira spp. By PCR, 2 of 23 (9.1%) kidney and 2 of 23 (9.1%) liver were positive for pathogenic Leptospira spp. Abdominal effusion from 4 dogs were PCR-positive for pathogenic Leptospira spp. The species detected were L. interrogans, L. borgpetersenii, L. kirschneri, and L. kmetyi by partial 16S rRNA sequencing. We further identified and characterized 11 Leptospira spp. isolates from 8 dogs as serovars Bataviae, Javanica, and Australis. The mortality rate of the Leptospira-infected dogs was high (18 of 53; 34%).

Seroprevalence and Distribution of Leptospiral Serovars in Livestock (cattle, Goats, and Sheep) in Flood-prone Kelantan, Malaysia.

INTRODUCTION: Leptospirosis is a bacterial disease that affects both humans and animals, the occurrence of which increases markedly during and after heavy rainfall and flooding. The aim of this study was to determine the serological prevalence of leptospiral infection in livestock after a voluminous flood in 10 districts of the Malaysian state of Kelantan. MATERIAL AND METHODS: In December 2014, Kelantan was hit by an extensive flood. A total of 1,728 serum samples were collected from livestock from the state, comprised of 1,024 from cattle, 366 from goats and 338 from sheep, and they were tested using the microscopic agglutination test (MAT). RESULTS: Altogether, 203 (11.75%; 203/1728; 95% CI: 10.20%-13.30%) of the tested sera were found to be positive serologically. Cattle had the highest prevalence of 14.16% (145/1024), while goats and sheep had 11.20% (41/366) and 5.03% (17/338) respectively. The most frequent serovars detected were Hardjo-bovis (3.70%; 64/1728), Hebdomadis (2.08%; 36/1728) and Pomona (1.04%; 18/1728). There was a statistically significant association (P < 0.05) between livestock that were exposed to the flood and seropositivity. CONCLUSION: This study showed that flood is a risk factor that can play a role in the epidemiology of leptospiral infection in livestock.

Antigenic distribution, pathological changes, antibody response and serological detection in non-pregnant goats following experimental infection by Brucella melitensis.

This study describes the pathological changes, antibody response, isolation and distribution patterns following exposure of non-pregnant goats to live Brucella melitensis. Eighteen healthy adult female goats were divided into two equal groups. Group 1 was infected via conjunctival sac with 109  cfu/ml of B. melitensis, while Group 2 was similarly exposed to sterile PBS. Serum and swabs from the eyes and vagina were collected at 5-day intervals. On days 15, 30 and 75 post-infection, 3 goats from each group were killed before the conjunctiva, ovary, oviduct, uterine horn, uterine body and vagina, the submandibular, prescapular and supramammary lymph nodes, the mammary gland, liver, spleen, urinary bladder and synovial membranes were collected for bacterial isolation and pathological study. Exposure of non-pregnant goats to B. melitensis did not produce clinical signs and gross lesions but produced mild necrosis and inflammation in the lymph nodes, the organs of reproductive tract, the mammary gland and urinary bladder. In general, microscopic lesions were most severe in the D75 goats, followed by D30 and D15 goats. Brucella melitensis was most frequent and significantly (p < .05) isolated from the D30 (64.4 ± 25.2%) and least from D15 goats (39.3 ± 26.0%) goats. The organs that were most frequently isolated were the uterus, followed by the mammary gland, supramammary lymph node and urinary bladder. Earliest isolation from the ocular swabs was on day 5, while the vaginal swabs were on day 20 post-infection. The antibody response showed first significant (p < .05) increase on day 15 and reached peak on day 45 post-infection, corresponding with the first detection of sero-converter goats by the RBPT at 15 days and by the CFT at 40 days post-infection. In conclusion, infected non-pregnant goats shed B. melitensis through the vagina by day 20. The sero-positive goats were detectable by RBPT after 15 days but by CFT after 40 days. Since both serological tests detected positive goats at different time period of infection, paired-serum samplings might reduce this discrepancy.

Pathological changes, distribution and detection of Brucella melitensis in foetuses of experimentally-infected does.

BACKGROUND: Brucellosis of goats is caused by Brucella melitensis. It is a re-emerging zoonotic disease in many countries due to transmission from domestic animals and wildlife such as ibex, deer and wild buffaloes. OBJECTIVE: To describe the pathological changes, identification and distribution of B. melitensis in foetuses of experimentally infected does. METHODS: Twelve female goats of approximately 90 days pregnant were divided into 4 groups. Group 1 was exposed intra-conjunctival to 100 µL of sterile PBS while goats of Groups 2, 3 and 4 were similarly exposed to 100 µL of an inoculum containing 109 CFU/mL of live B. melitensis. Goats of these groups were killed at 15, 30 and 60 days post-inoculation, respectively. Foetal fluid and tissues were collected for bacterial identification (using direct bacterial culture, PCR and immuno-peroxidase staining) and histopathological examination. RESULTS: Bilateral intra-conjunctival exposure of pregnant does resulted in in-utero infection of the foetuses. All full-term foetuses of group 4 were either aborted or stillborn, showing petechiations of the skin or absence of hair coat with subcutaneous oedema. The internal organs showed most severe lesions. Immune-peroxidase staining revealed antigen distribution in all organs that became most extensive in group 4. Brucella melitensis was successfully isolated from the stomach content, foetal fluid and various other organs. CONCLUSION: Vertical transmission of caprine brucellosis was evident causing mild to moderate lesions in different organs. The samples of choice for isolation and identification of B. melitensis are stomach content as well as liver and spleen tissue.

First report of pathogenic Leptospira spp. isolated from urine and kidneys of naturally infected cats.

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. Healthy cat, as a potential source of exposure to humans, are likely underestimated owing to the lack of overt clinical signs associated with Leptospira spp. infection in this species. The aim of the study was to determine the exposure, shedding, and carrier status of leptospires in shelter cats in Malaysia by using serological, molecular, and bacteriological methods. For this study, 82 healthy cats from two shelters were sampled. The blood, urine, and kidneys were tested using the microscopic agglutination test (MAT), polymerase chain reaction (PCR), and bacterial culture. On the basis of serological, molecular, and/or culture techniques, the total detection of leptospiral infection was 29.3% (n = 24/82). Through culture techniques, 16.7% (n = 4/24) of the cats that tested positive were carriers with positive kidney cultures, and one cat was culture positive for both urine and kidney. The Leptospira spp. isolates were identified as pathogenic L. interrogans serovar Bataviae through serological and molecular methods. Through serological techniques, 87.5% (n = 21/24) had positive antibody titers (100-1600) and most of the Bataviae serogroup (n = 19/21). Using PCR, 16.7% (n = 4/24) of cats were shown to have pathogenic Leptospira spp. DNA in their urine. Furthermore, three out of four culture positive cats were serology negative. The present study reports the first retrieval of pathogenic leptospires from urine and kidneys obtained from naturally infected cats. The results provide evidence of the potential role of naturally infected cats in the transmission of leptospires. Additionally, leptospiral infection occurs sub-clinically in cats. The culture isolation provides evidence that healthy cats could be reservoirs of leptospiral infection, and this information may promote the development of disease prevention strategies for the cat population.

Risk Factors and Prediction of Leptospiral Seropositivity Among Dogs and Dog Handlers in Malaysia.

This study determined the potential risk factors that may contribute to seropositivity among dogs and dog handlers from working dog and dog shelter institutions. Data was collected from dogs (n = 266) and dog handlers (n = 161) using a standardised guided questionnaire. Serum obtained from the dogs and dog handlers was tested using the microscopic agglutination test (MAT). A logistic regression analysis was used to predict leptospiral seropositivity of dogs and dog handlers based on potential risk factors. A total of 22.2% of dogs and 21.7% of dog handlers were seropositive. The significant predictors for the dogs' seropositivity were presence of rats (OR = 4.61 (95% CI: 1.05, 20.33), p = 0.043) and shared common area (OR = 5.12 (95% CI: 1.94, 13.46), p = 0.001) within the organisation. Significant predictor for dog handler seropositivity was contact time with the dogs of more than six hours/day (OR = 3.28 (95% CI: 1.28, 8.40), p = 0.013) after controlling for the effect of other risk factors such as small mammal contact, rat infestation at home, flooding at housing area (within three months) and urban locality. The exposure to various disease sources identified poses risk to dogs and dog handlers. Risk could be reduced with adequate application of protection at work while handling dogs and thus limiting contact with these sources and reducing exposure to infection.

Serological Detection of Anti-Leptospira Antibodies in Shelter Cats in Malaysia.

Leptospirosis is one of the most widespread zoonotic diseases and despite extensive research, there is still a paucity of information regarding this disease in cats. The aim of this study was to investigate the prevalence of leptospirosis among the shelter cat population in Malaysia and to determine the most common infective Leptospira serogroups among them. Blood samples were collected from a total of 110 cats from 4 different shelters. The sampled cats appeared healthy, with minimal evidence of feline upper respiratory disease. The Microscopic Agglutination Test was used to detect anti-Leptospira antibodies against 20 pathogenic serovars. Based on a cut-off antibody titer of ≥1:100, 20 of 110 sheltered cats, showed presence of anti-Leptospira antibodies against at least 1 serovar. The serodetection of leptospirosis was 18.18% (95% confidence interval 12.09-26.42). The most commonly detected serogroups were Bataviae, Javanica, and Ballum, with antibody titers ranging from 1:100 to 1:1600. Knowledge of the predominant infective serovars in hosts worldwide and regionally is imperative for understanding the epidemiology of this zoonotic disease. Serosurveillance is the first step in this process. Further studies are warranted for investigation of urinary shedding in naturally infected cats with leptospirosis, using Polymerase chain reaction (PCR) and organism isolation followed by serovars identification.

Staphylococcal cassette chromosome mec (SCCmec) and characterization of the attachment site (attB) of methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible Staphylococcus aureus (MSSA) isolates.

This study was designed to screen for SCCmec types and to characterize the attachment site (attB) and universal insertion site (orfX) of SCCmec in a collection of 27 isolates (n = 11) methicillin resistant S. aureus and (n = 16) methicillin susceptible S. aureus isolates in Malaysia. Screening of SCCmec types and characterization of the attachment site was carried out using PCR amplification and Sanger's sequencing method. The result showed that a large proportion of the MRSA isolates carried SCCmec type III 7/11 (63%). Three isolates 3/11 (27%) and 1/11 (9.0%) carried SCCmec type II and IVd respectively. Amplification of the universal insertion site of the SCCmec (orfX) and attachment site (attB) showed that all 16 S. aureus isolates were positive for the orfX gene, while only 7 were positive for the attB gene. Phylogenetic diversity showed that the isolates clustered around strains with features similar to a community acquired MRSA. In conclusion, a high carriage rate of SCCmec type III was observed. The result also showed that all the S. aureus isolates have the orfX structure; however, not all isolates possesses the attB site on the 3' end of the orfX region.

Retrospective Study of Leptospirosis in Malaysia.

Leptospirosis is a bacterial disease transmitted to humans and animals by direct or indirect contact with urine or body fluids from infected animals especially rodents. Infection can be associated with wide clinical spectrum varying from asymptomatic to severe multi-organ syndrome with life-threatening consequences. We conducted a review of published studies on incidences, case reports, sero-epidemiological surveys from year 2000 to 2015 using different electronic data bases. Our study revealed that majority of the studies were conducted in Peninsular Malaysia and predominantly among high-risk human groups. Most of the studies on domestic animals were conducted in the 1980s; hence, the current status of leptospirosis among domestic animal population remains largely unknown. There tend to be a sharp rise in incidence rate among human population in the year 2014 which was attributed to flooding and heavy rainfall experienced as well as recreational activities. Several gaps in epidemiological knowledge were also disclosed.

Prevalence of Escherichia Coli O157:H7 and Enterobacteriaceae on Hands of Workers in Halal Cattle Abattoirs in Peninsular Malaysia.

BACKGROUND: Several occupational diseases of multiple origins are encountered among abattoir workers. Presence of indicator microorganisms (coliforms) on hands of workers can be used a gauge for hygienic practices. METHODS: A cross-sectional study was performed to assess the prevalence of E.coli and enterobacteriaceae among Halal abattoir workers in some government halal abattoirs of Malaysia. A total of one hundred and sixty-five hand swab samples were collected from workers of Halal abattoirs in Malaysia. The samples were subjected to microbiological analysis for characterisation and serotyping. RESULTS: The results have shown that no Escherichia coli O157:H7 was isolated on the hands of abattoir workers before and after work. However, a total prevalence of 9.7% was recorded for all samples during work. For non-O157:H7, total prevalence of 33.3% during work and 13% after work were obtained. High prevalence was recorded in sample taken during work from Tampin, Jasin and Kemaman (100% each) while low prevalence where observed in Shah Alam, Banting and Ipoh (20% each). CONCLUSIONS: Based on the findings the hygienic practices of hand washing among the workers in few locations was found to be low especially after work.

Prevalence of Escherichia Coli O157:H7 and Enterobacteriaceae on Hands of Workers in Halal Cattle Abattoirs in Peninsular Malaysia

Background: Several occupational diseases of multiple origins are encountered among abattoir workers. Presence of indicator microorganisms (coliforms) on hands of workers can be used a gauge for hygienic practices. Methods: A cross-sectional study was performed to assess the prevalence of E.coli and enterobacteriaceae among Halal abattoir workers in some government halal abattoirs of Malaysia. A total of one hundred and sixty-five hand swab samples were collected from workers of Halal abattoirs in Malaysia. The samples were subjected to microbiological analysis for characterisation and serotyping. Results: The results have shown that no Escherichia coli O157:H7 was isolated on the hands of abattoir workers before and after work. However, a total prevalence of 9.7% was recorded for all samples during work. For non-O157:H7, total prevalence of 33.3% during work and 13% after work were obtained. High prevalence was recorded in sample taken during work from Tampin, Jasin and Kemaman (100% each) while low prevalence where observed in Shah Alam, Banting and Ipoh (20% each). Conclusions: Based on the findings the hygienic practices of hand washing among the workers in few locations was found to be low especially after work.

Serological diagnostic potential of recombinant outer membrane proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked immunosorbent assay.

BACKGROUND: Brucella melitensis is the most important pathogenic species of Brucella spp. which affects goats and sheep and causes caprine and ovine brucellosis, respectively. Serological tests for diagnosis of brucellosis such as Rose Bengal plate test (RBPT) and enzyme-linked immunosorbent assay (ELISA) usually utilize smooth lipopolysaccharides (S-LPS) as a diagnostic antigen which could give false positive serological reactions. Outer membrane proteins (OMP) of B. melitensis have been used as alternative diagnostic antigens rather than S-LPS for differential serological diagnosis of brucellosis, mainly in ELISA with single recombinant OMP (rOMP) as a diagnostic antigen. Nevertheless, the use of single format mainly showed lack of sensitivity against the desired rOMP. Therefore, this study aimed to determine whether a newly developed rOMPs indirect ELISA (rOMPs I-ELISA), based on combination of rOMP25, rOMP28 and rOMP31of B. melitensis, has a potential benefit for use in the serodiagnosis of brucellosis. METHODS: In this study, omp25, omp28 and omp31 of B. melitensis were cloned and expressed using prokaryotic pET-32 Ek/LIC system and their respective rOMPs were combined as one coating antigen to develop rOMPs I-ELISA. Three groups of BALB/c mice were used to elicit antibody response. Group 1, infected with B. melitensis strain 0331 field strain; group 2, injected with B. melitensis Rev.1 vaccine strain and group 3, infected with Yersinia enterocolitica O:9. Antibody responses in three groups of mice were investigated using Rose Bengal plate test (RBPT) and rOMPs I-ELISA. RESULTS: The production of rOMP25, rOMP28 and rOMP31 of B. melitensis were achieved and Western immunoblotting analysis demonstrated their reactivity. The RBPT was unable to differentiate the vaccinated mice (group 2) and mice infected with Y. enterocolitica O:9 (group 3) and categorized them wrongly as positive for brucellosis. In contrast, the rOMPs I-ELISA was able to differentiate the mice infected with B. melitensis strain 0331 (group 1) from both of group 2 and group 3, and recorded 100% sensitivity and 100% specificity. CONCLUSIONS: The results of this study suggested that rOMPs of B. melitensis has potential diagnostic ability to differentiate the FPSR in serological diagnosis of brucellosis.

A case-control study of risk factors for bovine brucellosis seropositivity in Peninsular Malaysia.

Bovine brucellosis was first reported in Peninsular Malaysia in 1950. A subsequent survey conducted in the country revealed that the disease was widespread. Current knowledge on the potential risk factors for brucellosis occurrence on cattle farms in Malaysia is lacking. Therefore, we conducted a case-control study to identify the potential herd-level risk factors for bovine brucellosis occurrence in four states in the country, namely Kelantan, Pahang, Selangor and Negeri Sembilan. Thirty-five cases and 36 controls of herds were selected where data on farm management, biosecurity, medical history and public health were collected. Multivariable logistic regression identified that Brucella seropositive herds were more likely to; have some interaction with wildlife (OR 8.9, 95% CI = 1.59-50.05); originated from farms where multiple species such as buffalo/others (OR 41.8, 95% CI = 3.94-443.19) and goat/sheep (OR 8.9, 95%Cl = 1.10-71.83) were reared, practice extensive production system (OR 13.6, 95% CI 1.31-140.24) and have had episodes of abortion in the past (OR 51.8, 95% CI = 4.54-590.90) when compared to seronegative herds. Considering the lack of information on the epidemiology of bovine brucellosis in peninsular Malaysia and absence of information on preventing the inception or spread of the disease, this report could contribute to the on-going area-wise national brucellosis eradication program.

Production and characterization of a polyclonal antibody of anti-rLipL21-IgG against leptospira for early detection of acute leptospirosis.

Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein of Leptospira interrogans serovar Pomona. We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection.

Case-control study on risk factors associated with Brucella melitensis in goat farms in Peninsular Malaysia.

Caprine brucellosis is a bacterial zoonotic infection affecting goats especially in developing countries all over the world. In Malaysia, the risk factors associated with this infection in farms have not been studied. A case-control study was carried out in goat farms in four states of Malaysia to elucidate the risk factors associated with the infection on the farms using structured questionnaires and face-to-face interviews. Results indicate that the introduction of new animals (OR = 5.25; 90 % CI = 1.46, 18.88), younger age category of farms (OR = 5.53; 90 % CI = 1.09, 21.66), and farms with single breed of goats (OR = 8.50; 90 % CI = 1.27, 41.97) were significant risk factors for brucellosis. In order to control brucellosis or possibly eradicate it in goat farms, these factors need to be dealt with. Enforcing stringent importation protocols or complete ban of goat importation from brucellosis endemic countries will help reduce risk of introducing new infection into the country.

Bovine brucellosis trends in Malaysia between 2000 and 2008.

BACKGROUND: Bovine brucellosis is an important disease affecting cattle characterised by abortion, still birth, reduced milk production, weak foetus and infertility in both males and females. There is wide distribution of the disease among cattle and several wildlife species. Bovine brucellosis is commonly caused by B. abortus and very occasionally B. melitensis and B. suis. The distribution of bovine brucellosis in cattle has not been described in Malaysia. In this paper we describe the distribution, pattern and trend of bovine brucellosis in Peninsular Malaysia between 2000 and 2008 based on serological data obtained from nationwide B. abortus serosurveillance activities in cattle populations. RESULTS: Brucella antibodies were detected in 21.8% of sampled herds (95% CI, 21.01-22.59) and 2.5% (95% CI; 2.45-2.55) of sampled cattle. The state of Pahang had the highest animal and herd-level seroprevalence of 5.3 and 43.6%, respectively. The herd-level seroprevalence varied but remained high (18-26%) over the period of study and generally increased from 2000 to 2008. Seropositive herds clustered around the central part of the peninsula within the period of the study. The months of September, October and November illustrated the highest rates with corresponding seroprevalences of 33.2, 38.4 and 33.9%, respectively. A noticeable variation was observed in the cattle-level seroprevalence, but the rate remained relatively low (<5%). The chi-square statistics showed herd size (χ2 = 1206.077, df = 2, p = 0.001), breed (χ2 = 37.429, df = 1, p = 0.001), month of sampling (χ2 = 51.596, df = 11 p = 0.001), year (χ2 = 40.08, df = 8, p = 0.001) and state (χ2 = 541.038, df = 10, p = 0.001) to be associated with increased seropositivity. CONCLUSION: Bovine brucellosis is widespread among herds in Peninsular Malaysia at a low within-herd seroprevalence rate.

Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia.

A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).