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Seroprevalence of Leptospirosis in Working Dogs
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Seroprevalence of Leptospirosis in working dogs S.F. Lau, J.Y. Wong, K.H. Khor, M.A. Roslan, M.S. Abdul Rahman, S.K. Bejo, R. Radzi, A.R. Bahaman
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S1938-9736(17)30103-4 https://doi.org/10.1053/j.tcam.2017.12.001 TCAM301
To appear in: Topics in Companion Animal Medicine Cite this article as: S.F. Lau, J.Y. Wong, K.H. Khor, M.A. Roslan, M.S. Abdul Rahman, S.K. Bejo, R. Radzi and A.R. Bahaman, Seroprevalence of Leptospirosis in working dogs, Topics in Companion Animal Medicine,doi:10.1053/j.tcam.2017.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Seroprevalence of Leptospirosis in working dogs
S.F. Lau
a1
, J.Y. Wong a, K.H. Khor a, M.A. Roslan b, M.S. Abdul Rahman b, S.K. Bejo b, R.
Radzi a, A.R. Bahaman b a
Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra
Malaysia, 43400 Serdang, Malaysia b
Department of Veterinary Pathology & Microbiology, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400 Serdang, Malaysia
1
Corresponding author
Seng Fong Lau, DVM, PhD Present address: Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Malaysia Tel.: +60386093927 E-mail address: [email protected] (S.F. Lau)
Abstract Working dogs are canine animals that have been trained to assist human beings in carrying out various tasks. They help in guarding property, performing rescues, assisting the visually impaired or physically handicapped, searching for drugs, explosives, and others. Leptospirosis is one of the most widespread zoonotic diseases in the world and a commonly occurring disease of the tropics and subtropics. In Malaysia, all working dogs are normally vaccinated with serovars, Pomona, Icterohaemorrhagiae, Canicola and Grippotyphosa based on protocols recommended from other countries. The duration of immunity in vaccinated dogs for Leptospira can last up to 13 months, however there is no full cross protection between the different serovars. Five representative canine units from different government agencies in Malaysia (n=96 dogs) were recruited in this study. For detection, the microscopic agglutination test was performed by incubating
the
serum
from
dogs
with
various
serovars
of
leptospires,
namely,
Icterohaemorrhagiae, Canicola, Pomona, Grippotyphosa, Australis, Bataviae, Javanica, Tarassovi, Hebdomadis, Lai and Pyrogenes. The plasma obtained was used for polymerase chain reaction (PCR) analysis, for the detection of 16S rRNA and lipL 32 genes of Leptospira. Out of the 96 dogs sampled, only three dogs were positive towards serovars, Australis, Bataviae and Javanica, based on the cut-off point at 1:80. The seroprevalence of canine leptospirosis in this population was 3.1% (n=3/96). However, all 96 blood samples of working dogs, tested negative for both pathogenic and non-pathogenic Leptospira genes. The results revealed that, by vaccination alone, working dogs were not fully protected against leptospirosis, and could pose a risk to dog handlers. A preventative and control protocol for leptospirosis is warranted, and its implementation should be monitored and improved accordingly from time to time, in order to maintain a healthy condition in both working dogs and their handlers.
Keywords: Working dogs, leptospirosis, vaccination, microscopic agglutination test, handlers
Introduction Working dogs are a group of canine animals trained to perform various tasks, to assist human beings such as, guard dogs trained to protect property, rescue dogs that are used in rescue operations and, search dogs to detect for drugs, explosives and others. Not only are they trained in protecting human beings but can also aid people with disabilities such as, blindness. Man’s oldest animal friend has come to help humans in remarkable ways. The handlers or caretakers of working dogs are very careful about the health and welfare of their dogs, and ensure their dog’s physical health is optimal. The human-animal bond is pivotal to the modern working dog team, and dogs provide much more than just work to their human partners [1]. Leptospirosis is one of the most widespread zoonotic diseases worldwide, commonly occurring in tropical and subtropical regions [2]. It produces a wide array of clinical signs in both human and animals, ranging from undifferentiated mild fever to multi-organ failure [3]. The clinical features of canine leptospirosis can either be subclinical or clinical [4,5]. Common clinical signs include lethargy, anorexia and vomiting, and the clinical signs are believed to be related to different serovars, geographic area, pathogenicity, and immunity of host. For example, dogs infected with serovar Pomona were more likely to suffer from vomiting due to renal failure [6]. Unfortunately, none of these symptoms is specific to the disease and diagnosis based solely on clinical signs is not sufficient. Therefore, accurate diagnosis is essential. Methods used for the diagnosis of leptospirosis includes, the microscopic agglutination test (MAT), polymerase chain reaction (PCR) technique, and enzyme-linked immunosorbent assay (ELISA) test kits. In animals, this disease is always underdiagnosed, especially in developing countries due to economic burden, lack of facilities, and cultural barriers [7,8]. The
first canine leptospirosis in Malaysia was reported in 1928, a Hebdomadis serovar [9]. Since the 1980s, 37 Leptospira serovars had been isolated from both animals and humans in Malaysia [10]. The most recent studies on the seroprevalence of canine leptospirosis in selected dog populations from Klang Valley, Malaysia revealed that the most common serovars were Canicola (15.8%, three out of 19 dogs with renal disease), Bataviae (3.8%, three out of 80 sheltered dogs) and Icterohaemorrhagiae (5.3%, one out of 19 healthy pet dogs) [8,11]. The evidence of seropositivity of leptospirosis among dog populations is of public health concern, due to the increasingly close contact between reservoir dogs and human pet-owners, veterinarians, animal handlers, and people employed in animal husbandry related jobs. Although the working dog population belongs to the high risk group, the seroprevalence of leptospirosis to the best of our knowledge, has never been investigated in this population before. This group of dogs are generally assumed to be well protected against leptospirosis, as they are vaccinated yearly. However, this population of dogs actually work in high risk environments, and the likelihood of them acquiring leptospirosis is in fact higher. Therefore, the current preliminary study may help in elucidating the current status of canine leptospirosis in the working dog population in Malaysia. This data would also provide veterinarians which a general estimation of the spatial distribution, and trend of this infectious disease among the working dog population, and to determine whether it is of public health concern especially to handlers. The objectives of this study are, to investigate the prevalence of canine leptospirosis among the working dog population in Malaysia and, to determine the most common Leptospira serovar in infected dogs. From the results obtained, appropriate preventive measures can be recommended for the future.
Materials and methods Blood sample collection Five representative canine units from different government agencies in Malaysia were recruited in this study. Before commencing the study, approval from both the Institutional Animal Care and Use Committee (UPM/IACUC/FYP.2015/FPV.005) and government agencies, were obtained. The dogs were manually restrained by the handlers or caretakers from the canine units for blood collection. Approximately 3-5ml of blood was collected via cephalic or saphenous venipuncture. Blood samples obtained were immediately transferred into plain and Ethylenediaminetetraacetic Acid (EDTA) tubes. Information of the working dogs such as name, sex, breed, age, vaccination status and medical history was recorded. All blood samples were stored in polystyrene ice boxes with ice packs, and immediately transported to the Bacteriology Laboratory in Faculty of Veterinary Medicine, University Putra Malaysia. The blood samples in plain tubes were immediately centrifuged at 4000 rpm for 5 minutes. After centrifugation, both serum and plasma samples were isolated and transferred into 1.5ml Eppendorf tubes and then stored at -20°C for further analysis.
Microscopic Agglutination Test (MAT) Eleven serovars of live leptospires were subcultured in Ellinhausen-McCulloughJohnson-Harris (EMJH) medium, and incubated at 30°C for 5-7 days. The serovars tested included, Icterohaemorrhagiae, Canicola, Pomona, Grippotyphosa, Australis, Bataviae, Javanica, Tarassovi, Hebdomadis, Lai and Pyrogenes. These live leptospires were examined under a dark microscopic before application for the MAT.
Serial dilutions of the sera samples, negative and positive controls were prepared in the sterile microtitre plate wells. A total of 14 samples were loaded into each microtire plate, including a positive and negative control, and a serial dilution up to 1:640. All wells were filled with 50µl of phosphate buffer saline (PBS). Hyperimmune serum was used as a positive control. Serial dilutions were carried out by pipetting 50µl starting from the 1st column until 12th column. The last dilution was 1:640 and the 50µl dilution was discarded. These steps were repeated for all the serum samples. In total, 50µl of live leptospiral solutions were added to all wells and, mixed thoroughly using an incubator shaker for five minutes and subsequently incubated at 37.1°C for two hours. After incubation, all the sera sample solutions were placed onto glass slides and examined under a dark field microscope. Any evidence of microscopic agglutination was determined until the highest antibody titre for each serum sample. The cut-off point was determined at 1:80. If >50% agglutination was detected at this cut-off point or more, it was recorded as positive.
Polymerase Chain Reaction (PCR) DNA extraction from blood plasma was carried out using the QIAamp DNA blood Mini Kits (QIAGEN, Germany) according to the methods described in a previous study [11]. PCR reaction mixtures were prepared using the Top Taq Master Mix cocktail solution. The PCR cocktail solution consisted of, 12.5µl of Top Taq Master Mix 2x, 1.25µl of Genus Specific Forward Primer (16’S rRNA; 5’-CAT GCA AGT CAA GCG GAG TA-3’), 12.5µl of Genus Specific Reverse Primer (16’S rRNA; 5’-AGT TGA GCC CGC AGT TTT C-3’ ), 1.25µl of Pathogenic Specific Forward Primer (lipL 32; 5’-GTC GAC ATG AAA AAA CTT TCG ATT TTG-3’), 1.25µl of Pathogenic Specific Reverse Primer (lipL 32; 5’-CTG CAG TTA CTT AGT CGC
GTC AGA AGC-3’) and 2.5µl of RNase-free water. Each PCR tube contained 20µl of PCR cocktail solution and 5µl of sample DNA template. The mixture was vortexed followed by a short spin step. PCR amplification was carried out in a Master cycler Pro S PCR machine (Eppendorf, Germany). The amplified PCR products were electrophoresed on 1.5% agarose gels. A total of 2µl of 100bp DNA ladder each, was loaded in the first and final wells. Then, 5µl of PCR product and 2µl of loading dye was placed in each well. Electrophoresis was carried out at 100V for one hour and 45 minutes. After electrophoresis, the agarose gels were stained with ethidium bromide solution for 5 minutes and then de-stained in distilled water for 5 minutes. The agarose gels were viewed under ultraviolet light using a transilluminator.
Results A total of 96 working dogs were selected from five canine units, as a representative sample of the approximate 300 total working dog population in Malaysia. All the selected working dogs’ vaccination status was up to date. Breeds of the working dogs included, Labrador, Malinois, German Shepherd, Belgian Shepherd, Rottweiler and English Springer Spaniel. Out of 96 dogs, 69% were male whereas 31% were female dogs. The age range was between one yearold to 13 years-old (Mean= 4 years-old). Annual vaccines were administered to protect against four leptospiral serovars namely, Icterohaemorrhagiae, Canicola, Pomona and Grippotyphosa serovars (VANGUARD Plus 5 L4, Pfizer Inc., USA).
Microscopic Agglutination Test (MAT) Of the samples tested, only three dog sera samples were considered positive towards the Australis, Bataviae and Javanica serovars, based on the cut-off point at 1:80. The seroprevalence of canine leptospirosis in this population was 3.1% (n=96). The dog that tested positive against serovar Australis (1:80), was a 9-year-old male Labrador which received its last vaccination seven months ago. This particular dog survived leptospirosis in 2013 and since then, the annual check-ups revealed no abnormalities. The Bataviae serovar (1:80) tested positive in a 4-year-old male German Shepherd that received its last vaccination a year ago, whereas the serovar Javanica (1: 160) tested positive in a 6-year-old female German Shepherd dog, vaccinated 3 months ago. Both dogs were healthy with no previous record of canine leptospirosis. Three of the positive dogs were middle-aged dogs and both sex included. Eight other dogs tested seropositive against serovars, Icterohaemorrhagiae, Pomona, Australis, Bataviae, Javanica, and Lai with low titers (<1:80; Table 1). Thus, these dogs were considered negative.
Polymerase Chain Reaction (PCR) Through PCR detection of the Leptospira gene, all the 96 blood samples of working dogs were negative for both pathogenic and non-pathogenic Leptospira strains.
Discussion Dogs are a good indicator of the distribution of different leptospiral serovars in nature. Therefore, occasional serological screening of dogs is warranted, to detect possible serovar changes and its distribution, despite the fact that the prevalence of canine leptospirosis has decreased worldwide, with the use of bivalent vaccines [12,13]. Various countries have reported
the re-emergence of canine leptospirosis, mostly due to changes in the infecting serovars in the last decade, indicative that, most of the infections were caused by Leptospira serovars which are not covered by the vaccine [13,14]. In Malaysia, there is a paucity of information regarding the current circulating serovars, among the dog population. Most of the dogs are vaccinated with Pomona, Icterohaemorrhagiae, Canicola and Grippotyphosa serovars, based on a protocol adopted from other countries. A vaccination protocol should be based on the major serovars causing infection, in a local setting. Different serovars are known to adapt to specific reservoir or maintenance hosts, and vary according to the geographical location [15]. Dogs in Europe are potentially exposed to the serogroups, Icterohaemorrhagiae (maintenance host = rat), Australis (maintenance host = wildlife), Canicola (maintenance host = dog), Grippotyphosa and Sejroe (maintenance host for both serogroups = rodents). The incidence of the latter two groups varies with the presence of the maintenance host [15]. More recent studies in Germany [16] and Greece [17] have identified antibodies against the serogroup, Pomona in ill dogs. Ward [18] reported a higher incidence of leptospirosis in middle-aged male dogs (between 4 to 10-year-old). He suggested that, male, middle-aged dogs and large-breed dogs were usually more active outdoors, and this could increase their risk to exposure to Leptospira serovars. Working dogs as compared to the pet dog population, have a higher risk of infection to leptospirosis. All the seropositive dogs belonged to the search and rescue group that work outdoors. Their jobs include, searching for lost victims in the forest, walking along riverbanks, damp soil, sniffling cadavers, rescuing victims trapped in collapsed soil structures and landslides as well as others. All of these activities can potentially expose working dogs to water and soil contaminated with Leptospira. Leptospires is known to survive for several months in the environment under favorable conditions [19]. One of the working dog units in this study reported
an outbreak of leptospirosis in 2013. One survivor tested positive using MAT (1:80), for serovar Australis and might act as carrier. As a limitation in this study, the sera samples were only analysed in one laboratory and no follow up had been done in all these three dogs, which were considered positive based on the cut-off point at 1:80. Hence, in the current study, a positive MAT result was indicative of the presence of circulating antibodies against Leptospira serovar, either from a recent natural infection, past exposure or recent vaccination. Current diagnostic tools available for leptospirosis include, direct dark-field microscopy examination of blood, bacteriological culturing, polymerase chain reaction (PCR), isothermal methods, IgM or IgG enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, macroscopic or rapid slide agglutination test (MSAT or RSAT), indirect hemagglutination assay (IHA), complement fixation test (CF) and lateral flow assay (LFA) [20, 21]. Selecting a diagnostic test depends on several factors such as, diagnostic accuracy (sensitivity, specificity and predictive value), financial feasibility, technical or practical feasibility, and whether early or rapid results are required [21]. Both the leptospiral culture and MAT, are still used as the gold standard for leptospirosis diagnosis. Leptospiral culture is less practical in clinical diagnostic, when rapid results are required, as Leptospires are slow-growing bacteria, and require longer time for culturing. MAT however, is used to assess the antibody titers of serum samples against Leptospira from different serovar groups. [22]. It has been recommended that, a paired serum sample at 14 days interval be used, as a four-fold or higher antibody titer will help in diagnosing leptospirosis [23]. The cut off antibody titers used in different countries varies, ranging from 1:20 to 1:3200, depending on the condition of infection, or whether Leptospirosis is endemic in the country [24,25]. The MAT however, is not useful for early diagnosis as anti-Leptospira antibodies can only be detected by the second week after exposure to infection. Other limitations
of MAT is that it is time consuming, can be potentially hazardous to laboratory workers, requires well-trained personnel and repeated sub-culturing of many strains of Leptospira [21]. Both MAT and ELISA cannot be used to differentiate between vaccine antibodies and natural infection. In addition to that, vaccinated dogs developed variable seroconversion after vaccination and there were no correlation between post-vaccination titers and protection. Previous study reported dogs vaccinated with a bivalent vaccine developed MAT titers <1:80, which lasted only one to four months but in another study, dogs vaccinated with quadrivalent vaccine developed MAT titers >1: 1600. Hence, there is an urgent need to improve current diagnostic methods for investigating leptospirosis and differentiating vaccine antibodies and natural infection [26, 27, 28, 29]. A low titer (<1:80) against different Leptospira serovars was detected in eight dogs. However, healthy dogs with detectable levels of antibodies must be interpreted carefully, and the interpretation of the results highly depends upon the endemicity of the disease in an area as well as the vaccination history. The possibility of previous exposure in this group of dogs cannot be ruled out either. The lower titers can be interpreted as antibody cross-reactivity between serovars [30]. Cross-reaction between leptospires of the same serogroup might occur, as reported in a previous study of seroprevalence of cattle leptospirosis. The authors reported, infection by L. balcanica, that lead to a false positive L. hardjo reaction [31]. According to Gautum [25], crossreaction between Leptospira serovars could occur due to the production of both IgM and IgG antibodies that can agglutinate to multiple leptospira serovars. Cross protection with the use of commercial vaccines for Canicola and Icterohaemorrhagiae serovars has been reported, but not for other serovars [32]. Therefore, a low cut-off antibody titer used in MAT may overestimate seroprevalence of canine leptospirosis and the prevalence of clinical leptospirosis. According to
Klaasen [33] and Schreiber [34], the duration of immunity of vaccinated dogs for Leptospira, can last up to 13 months, but is not necessarily associated with the presence of circulating antiLeptospira antibodies, 15 weeks post-vaccination. The vaccinated dogs were protected from leptospiral infection despite of its low or even undetectable levels of agglutinating serum antibodies [33]. The PCR technique was used to amplify leptospiral DNA, for the detection in blood and urine samples. It is important to detect pathogenic Leptospira genes such as, LipL32, Lig A and Lig B, and is effective for early diagnosis with clinical symptoms [35]. According to Harkin [36], the sensitivity and specificity to detect Leptospira genes from urine samples through PCR, can be as high as 100% and 88.3%, respectively. In contrast, the PCR assay has a 90% sensitivity in the first five days of illness, and greatly decreases to 50% if blood samples are used [37]. Since the dogs in this study all appeared healthy with no clinical signs, the negative PCR results were not unexpected. Urine samples should be used in the future study as it has higher sensitivity in comparison to the blood samples through PCR. Due to the widespread nature of this zoonotic disease, Leptospirosis is of public health concern. Therefore, it is important to determine the serovars that infect the dog population, as office handlers or care takers are constantly in contact with these animals. According to Masuzawa [38], the seroprevalence of L. australis and L. javanica among human beings in Phillipines was 8.5% and 1.4% (n=71), respectively. In addition, the seroprevalence of L. australis and L. javanica has also been detected among dairy farm workers in India, which was approximately 12.8% and 17.9% (n=41), respectively [39]. L. batavia was also reported causing tubulointerstitial renal failure in two children [40]. The seropositive results found in the current study, among the working dog population, suggests that humans are potentially at risk of
exposure, and the possibility of direct or indirect transmission of leptospirosis from dogs to human, cannot be ruled out. In addition to the routine annual vaccination, chemoprophylasis using doxycycline, might be useful in preventing and controlling canine leptospirosis among working dogs. The advantages of this preventive measure has been reported by the U.S. military [41]. Strict kennel sanitation, isolation of infected dogs and rodent control, are appropriate strategies to reduce the spread of leptospirosis. Routine herd screening of leptospirosis using MAT and PCR assay for working dogs, should be conducted to monitor the emergence of other serovars, and to provide early diagnosis for immediate and appropriate antimicrobial treatment.
Conclusion The seroprevalence of canine leptospirosis among the working dog population in Malaysia was 3.1% (n=3/96). The pathogenic infecting serovars detected include, L. australis, L. bataviae and L. javanica. To the best of our knowledge, this study is the first to report the seroprevalence of leptospirosis in working dogs from Malaysia. Effective preventative and control protocols for leptospirosis, should be implemented in order to maintain a healthy condition in both dogs and handlers.
Acknowledgements The authors would like to thank the staff from the Bacteriology Laboratory of the Faculty of Veterinary Medicine, Universiti Putra Malaysia for their assistance in processing the samples and technical help.
Table 1 List of the working dogs that showed antibody-antigen reaction to form agglutination during the microscopic agglutination test. Breed
Gender
Age (year)
Serovar
Antibody titer
Female
6
Javanica
1:160
Labrador
Male
9
Australis
1:80
German Shepherd
Male
4
Bataviae
1:80
German Shepherd
Female
6
Javanica
1:40
Labrador
Male
4
Icterohaemorrhagiae
1:40
Labrador
Male
7
Icterohaemorrhagiae
1:40
German Shepherd
Female
8
Lai
1:40
German Shepherd
Male
4
Pomona
1:20
Belgian Shepherd
Male
2
Hebdomadis
1:20
Labrador
Male
2
Javanica
1:20
German Shepherd
Male
3
Australis
1:20
German Shepherd
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Seroprevalence of Leptospirosis in working dogs S.F. Lau, J.Y. Wong, K.H. Khor, M.A. Roslan, M.S. Abdul Rahman, S.K. Bejo, R. Radzi, A.R. Bahaman
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To appear in: Topics in Companion Animal Medicine Cite this article as: S.F. Lau, J.Y. Wong, K.H. Khor, M.A. Roslan, M.S. Abdul Rahman, S.K. Bejo, R. Radzi and A.R. Bahaman, Seroprevalence of Leptospirosis in working dogs, Topics in Companion Animal Medicine,doi:10.1053/j.tcam.2017.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Seroprevalence of Leptospirosis in working dogs
S.F. Lau
a1
, J.Y. Wong a, K.H. Khor a, M.A. Roslan b, M.S. Abdul Rahman b, S.K. Bejo b, R.
Radzi a, A.R. Bahaman b a
Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra
Malaysia, 43400 Serdang, Malaysia b
Department of Veterinary Pathology & Microbiology, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400 Serdang, Malaysia
1
Corresponding author
Seng Fong Lau, DVM, PhD Present address: Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Malaysia Tel.: +60386093927 E-mail address: [email protected] (S.F. Lau)
Abstract Working dogs are canine animals that have been trained to assist human beings in carrying out various tasks. They help in guarding property, performing rescues, assisting the visually impaired or physically handicapped, searching for drugs, explosives, and others. Leptospirosis is one of the most widespread zoonotic diseases in the world and a commonly occurring disease of the tropics and subtropics. In Malaysia, all working dogs are normally vaccinated with serovars, Pomona, Icterohaemorrhagiae, Canicola and Grippotyphosa based on protocols recommended from other countries. The duration of immunity in vaccinated dogs for Leptospira can last up to 13 months, however there is no full cross protection between the different serovars. Five representative canine units from different government agencies in Malaysia (n=96 dogs) were recruited in this study. For detection, the microscopic agglutination test was performed by incubating
the
serum
from
dogs
with
various
serovars
of
leptospires,
namely,
Icterohaemorrhagiae, Canicola, Pomona, Grippotyphosa, Australis, Bataviae, Javanica, Tarassovi, Hebdomadis, Lai and Pyrogenes. The plasma obtained was used for polymerase chain reaction (PCR) analysis, for the detection of 16S rRNA and lipL 32 genes of Leptospira. Out of the 96 dogs sampled, only three dogs were positive towards serovars, Australis, Bataviae and Javanica, based on the cut-off point at 1:80. The seroprevalence of canine leptospirosis in this population was 3.1% (n=3/96). However, all 96 blood samples of working dogs, tested negative for both pathogenic and non-pathogenic Leptospira genes. The results revealed that, by vaccination alone, working dogs were not fully protected against leptospirosis, and could pose a risk to dog handlers. A preventative and control protocol for leptospirosis is warranted, and its implementation should be monitored and improved accordingly from time to time, in order to maintain a healthy condition in both working dogs and their handlers.
Keywords: Working dogs, leptospirosis, vaccination, microscopic agglutination test, handlers
Introduction Working dogs are a group of canine animals trained to perform various tasks, to assist human beings such as, guard dogs trained to protect property, rescue dogs that are used in rescue operations and, search dogs to detect for drugs, explosives and others. Not only are they trained in protecting human beings but can also aid people with disabilities such as, blindness. Man’s oldest animal friend has come to help humans in remarkable ways. The handlers or caretakers of working dogs are very careful about the health and welfare of their dogs, and ensure their dog’s physical health is optimal. The human-animal bond is pivotal to the modern working dog team, and dogs provide much more than just work to their human partners [1]. Leptospirosis is one of the most widespread zoonotic diseases worldwide, commonly occurring in tropical and subtropical regions [2]. It produces a wide array of clinical signs in both human and animals, ranging from undifferentiated mild fever to multi-organ failure [3]. The clinical features of canine leptospirosis can either be subclinical or clinical [4,5]. Common clinical signs include lethargy, anorexia and vomiting, and the clinical signs are believed to be related to different serovars, geographic area, pathogenicity, and immunity of host. For example, dogs infected with serovar Pomona were more likely to suffer from vomiting due to renal failure [6]. Unfortunately, none of these symptoms is specific to the disease and diagnosis based solely on clinical signs is not sufficient. Therefore, accurate diagnosis is essential. Methods used for the diagnosis of leptospirosis includes, the microscopic agglutination test (MAT), polymerase chain reaction (PCR) technique, and enzyme-linked immunosorbent assay (ELISA) test kits. In animals, this disease is always underdiagnosed, especially in developing countries due to economic burden, lack of facilities, and cultural barriers [7,8]. The
first canine leptospirosis in Malaysia was reported in 1928, a Hebdomadis serovar [9]. Since the 1980s, 37 Leptospira serovars had been isolated from both animals and humans in Malaysia [10]. The most recent studies on the seroprevalence of canine leptospirosis in selected dog populations from Klang Valley, Malaysia revealed that the most common serovars were Canicola (15.8%, three out of 19 dogs with renal disease), Bataviae (3.8%, three out of 80 sheltered dogs) and Icterohaemorrhagiae (5.3%, one out of 19 healthy pet dogs) [8,11]. The evidence of seropositivity of leptospirosis among dog populations is of public health concern, due to the increasingly close contact between reservoir dogs and human pet-owners, veterinarians, animal handlers, and people employed in animal husbandry related jobs. Although the working dog population belongs to the high risk group, the seroprevalence of leptospirosis to the best of our knowledge, has never been investigated in this population before. This group of dogs are generally assumed to be well protected against leptospirosis, as they are vaccinated yearly. However, this population of dogs actually work in high risk environments, and the likelihood of them acquiring leptospirosis is in fact higher. Therefore, the current preliminary study may help in elucidating the current status of canine leptospirosis in the working dog population in Malaysia. This data would also provide veterinarians which a general estimation of the spatial distribution, and trend of this infectious disease among the working dog population, and to determine whether it is of public health concern especially to handlers. The objectives of this study are, to investigate the prevalence of canine leptospirosis among the working dog population in Malaysia and, to determine the most common Leptospira serovar in infected dogs. From the results obtained, appropriate preventive measures can be recommended for the future.
Materials and methods Blood sample collection Five representative canine units from different government agencies in Malaysia were recruited in this study. Before commencing the study, approval from both the Institutional Animal Care and Use Committee (UPM/IACUC/FYP.2015/FPV.005) and government agencies, were obtained. The dogs were manually restrained by the handlers or caretakers from the canine units for blood collection. Approximately 3-5ml of blood was collected via cephalic or saphenous venipuncture. Blood samples obtained were immediately transferred into plain and Ethylenediaminetetraacetic Acid (EDTA) tubes. Information of the working dogs such as name, sex, breed, age, vaccination status and medical history was recorded. All blood samples were stored in polystyrene ice boxes with ice packs, and immediately transported to the Bacteriology Laboratory in Faculty of Veterinary Medicine, University Putra Malaysia. The blood samples in plain tubes were immediately centrifuged at 4000 rpm for 5 minutes. After centrifugation, both serum and plasma samples were isolated and transferred into 1.5ml Eppendorf tubes and then stored at -20°C for further analysis.
Microscopic Agglutination Test (MAT) Eleven serovars of live leptospires were subcultured in Ellinhausen-McCulloughJohnson-Harris (EMJH) medium, and incubated at 30°C for 5-7 days. The serovars tested included, Icterohaemorrhagiae, Canicola, Pomona, Grippotyphosa, Australis, Bataviae, Javanica, Tarassovi, Hebdomadis, Lai and Pyrogenes. These live leptospires were examined under a dark microscopic before application for the MAT.
Serial dilutions of the sera samples, negative and positive controls were prepared in the sterile microtitre plate wells. A total of 14 samples were loaded into each microtire plate, including a positive and negative control, and a serial dilution up to 1:640. All wells were filled with 50µl of phosphate buffer saline (PBS). Hyperimmune serum was used as a positive control. Serial dilutions were carried out by pipetting 50µl starting from the 1st column until 12th column. The last dilution was 1:640 and the 50µl dilution was discarded. These steps were repeated for all the serum samples. In total, 50µl of live leptospiral solutions were added to all wells and, mixed thoroughly using an incubator shaker for five minutes and subsequently incubated at 37.1°C for two hours. After incubation, all the sera sample solutions were placed onto glass slides and examined under a dark field microscope. Any evidence of microscopic agglutination was determined until the highest antibody titre for each serum sample. The cut-off point was determined at 1:80. If >50% agglutination was detected at this cut-off point or more, it was recorded as positive.
Polymerase Chain Reaction (PCR) DNA extraction from blood plasma was carried out using the QIAamp DNA blood Mini Kits (QIAGEN, Germany) according to the methods described in a previous study [11]. PCR reaction mixtures were prepared using the Top Taq Master Mix cocktail solution. The PCR cocktail solution consisted of, 12.5µl of Top Taq Master Mix 2x, 1.25µl of Genus Specific Forward Primer (16’S rRNA; 5’-CAT GCA AGT CAA GCG GAG TA-3’), 12.5µl of Genus Specific Reverse Primer (16’S rRNA; 5’-AGT TGA GCC CGC AGT TTT C-3’ ), 1.25µl of Pathogenic Specific Forward Primer (lipL 32; 5’-GTC GAC ATG AAA AAA CTT TCG ATT TTG-3’), 1.25µl of Pathogenic Specific Reverse Primer (lipL 32; 5’-CTG CAG TTA CTT AGT CGC
GTC AGA AGC-3’) and 2.5µl of RNase-free water. Each PCR tube contained 20µl of PCR cocktail solution and 5µl of sample DNA template. The mixture was vortexed followed by a short spin step. PCR amplification was carried out in a Master cycler Pro S PCR machine (Eppendorf, Germany). The amplified PCR products were electrophoresed on 1.5% agarose gels. A total of 2µl of 100bp DNA ladder each, was loaded in the first and final wells. Then, 5µl of PCR product and 2µl of loading dye was placed in each well. Electrophoresis was carried out at 100V for one hour and 45 minutes. After electrophoresis, the agarose gels were stained with ethidium bromide solution for 5 minutes and then de-stained in distilled water for 5 minutes. The agarose gels were viewed under ultraviolet light using a transilluminator.
Results A total of 96 working dogs were selected from five canine units, as a representative sample of the approximate 300 total working dog population in Malaysia. All the selected working dogs’ vaccination status was up to date. Breeds of the working dogs included, Labrador, Malinois, German Shepherd, Belgian Shepherd, Rottweiler and English Springer Spaniel. Out of 96 dogs, 69% were male whereas 31% were female dogs. The age range was between one yearold to 13 years-old (Mean= 4 years-old). Annual vaccines were administered to protect against four leptospiral serovars namely, Icterohaemorrhagiae, Canicola, Pomona and Grippotyphosa serovars (VANGUARD Plus 5 L4, Pfizer Inc., USA).
Microscopic Agglutination Test (MAT) Of the samples tested, only three dog sera samples were considered positive towards the Australis, Bataviae and Javanica serovars, based on the cut-off point at 1:80. The seroprevalence of canine leptospirosis in this population was 3.1% (n=96). The dog that tested positive against serovar Australis (1:80), was a 9-year-old male Labrador which received its last vaccination seven months ago. This particular dog survived leptospirosis in 2013 and since then, the annual check-ups revealed no abnormalities. The Bataviae serovar (1:80) tested positive in a 4-year-old male German Shepherd that received its last vaccination a year ago, whereas the serovar Javanica (1: 160) tested positive in a 6-year-old female German Shepherd dog, vaccinated 3 months ago. Both dogs were healthy with no previous record of canine leptospirosis. Three of the positive dogs were middle-aged dogs and both sex included. Eight other dogs tested seropositive against serovars, Icterohaemorrhagiae, Pomona, Australis, Bataviae, Javanica, and Lai with low titers (<1:80; Table 1). Thus, these dogs were considered negative.
Polymerase Chain Reaction (PCR) Through PCR detection of the Leptospira gene, all the 96 blood samples of working dogs were negative for both pathogenic and non-pathogenic Leptospira strains.
Discussion Dogs are a good indicator of the distribution of different leptospiral serovars in nature. Therefore, occasional serological screening of dogs is warranted, to detect possible serovar changes and its distribution, despite the fact that the prevalence of canine leptospirosis has decreased worldwide, with the use of bivalent vaccines [12,13]. Various countries have reported
the re-emergence of canine leptospirosis, mostly due to changes in the infecting serovars in the last decade, indicative that, most of the infections were caused by Leptospira serovars which are not covered by the vaccine [13,14]. In Malaysia, there is a paucity of information regarding the current circulating serovars, among the dog population. Most of the dogs are vaccinated with Pomona, Icterohaemorrhagiae, Canicola and Grippotyphosa serovars, based on a protocol adopted from other countries. A vaccination protocol should be based on the major serovars causing infection, in a local setting. Different serovars are known to adapt to specific reservoir or maintenance hosts, and vary according to the geographical location [15]. Dogs in Europe are potentially exposed to the serogroups, Icterohaemorrhagiae (maintenance host = rat), Australis (maintenance host = wildlife), Canicola (maintenance host = dog), Grippotyphosa and Sejroe (maintenance host for both serogroups = rodents). The incidence of the latter two groups varies with the presence of the maintenance host [15]. More recent studies in Germany [16] and Greece [17] have identified antibodies against the serogroup, Pomona in ill dogs. Ward [18] reported a higher incidence of leptospirosis in middle-aged male dogs (between 4 to 10-year-old). He suggested that, male, middle-aged dogs and large-breed dogs were usually more active outdoors, and this could increase their risk to exposure to Leptospira serovars. Working dogs as compared to the pet dog population, have a higher risk of infection to leptospirosis. All the seropositive dogs belonged to the search and rescue group that work outdoors. Their jobs include, searching for lost victims in the forest, walking along riverbanks, damp soil, sniffling cadavers, rescuing victims trapped in collapsed soil structures and landslides as well as others. All of these activities can potentially expose working dogs to water and soil contaminated with Leptospira. Leptospires is known to survive for several months in the environment under favorable conditions [19]. One of the working dog units in this study reported
an outbreak of leptospirosis in 2013. One survivor tested positive using MAT (1:80), for serovar Australis and might act as carrier. As a limitation in this study, the sera samples were only analysed in one laboratory and no follow up had been done in all these three dogs, which were considered positive based on the cut-off point at 1:80. Hence, in the current study, a positive MAT result was indicative of the presence of circulating antibodies against Leptospira serovar, either from a recent natural infection, past exposure or recent vaccination. Current diagnostic tools available for leptospirosis include, direct dark-field microscopy examination of blood, bacteriological culturing, polymerase chain reaction (PCR), isothermal methods, IgM or IgG enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, macroscopic or rapid slide agglutination test (MSAT or RSAT), indirect hemagglutination assay (IHA), complement fixation test (CF) and lateral flow assay (LFA) [20, 21]. Selecting a diagnostic test depends on several factors such as, diagnostic accuracy (sensitivity, specificity and predictive value), financial feasibility, technical or practical feasibility, and whether early or rapid results are required [21]. Both the leptospiral culture and MAT, are still used as the gold standard for leptospirosis diagnosis. Leptospiral culture is less practical in clinical diagnostic, when rapid results are required, as Leptospires are slow-growing bacteria, and require longer time for culturing. MAT however, is used to assess the antibody titers of serum samples against Leptospira from different serovar groups. [22]. It has been recommended that, a paired serum sample at 14 days interval be used, as a four-fold or higher antibody titer will help in diagnosing leptospirosis [23]. The cut off antibody titers used in different countries varies, ranging from 1:20 to 1:3200, depending on the condition of infection, or whether Leptospirosis is endemic in the country [24,25]. The MAT however, is not useful for early diagnosis as anti-Leptospira antibodies can only be detected by the second week after exposure to infection. Other limitations
of MAT is that it is time consuming, can be potentially hazardous to laboratory workers, requires well-trained personnel and repeated sub-culturing of many strains of Leptospira [21]. Both MAT and ELISA cannot be used to differentiate between vaccine antibodies and natural infection. In addition to that, vaccinated dogs developed variable seroconversion after vaccination and there were no correlation between post-vaccination titers and protection. Previous study reported dogs vaccinated with a bivalent vaccine developed MAT titers <1:80, which lasted only one to four months but in another study, dogs vaccinated with quadrivalent vaccine developed MAT titers >1: 1600. Hence, there is an urgent need to improve current diagnostic methods for investigating leptospirosis and differentiating vaccine antibodies and natural infection [26, 27, 28, 29]. A low titer (<1:80) against different Leptospira serovars was detected in eight dogs. However, healthy dogs with detectable levels of antibodies must be interpreted carefully, and the interpretation of the results highly depends upon the endemicity of the disease in an area as well as the vaccination history. The possibility of previous exposure in this group of dogs cannot be ruled out either. The lower titers can be interpreted as antibody cross-reactivity between serovars [30]. Cross-reaction between leptospires of the same serogroup might occur, as reported in a previous study of seroprevalence of cattle leptospirosis. The authors reported, infection by L. balcanica, that lead to a false positive L. hardjo reaction [31]. According to Gautum [25], crossreaction between Leptospira serovars could occur due to the production of both IgM and IgG antibodies that can agglutinate to multiple leptospira serovars. Cross protection with the use of commercial vaccines for Canicola and Icterohaemorrhagiae serovars has been reported, but not for other serovars [32]. Therefore, a low cut-off antibody titer used in MAT may overestimate seroprevalence of canine leptospirosis and the prevalence of clinical leptospirosis. According to
Klaasen [33] and Schreiber [34], the duration of immunity of vaccinated dogs for Leptospira, can last up to 13 months, but is not necessarily associated with the presence of circulating antiLeptospira antibodies, 15 weeks post-vaccination. The vaccinated dogs were protected from leptospiral infection despite of its low or even undetectable levels of agglutinating serum antibodies [33]. The PCR technique was used to amplify leptospiral DNA, for the detection in blood and urine samples. It is important to detect pathogenic Leptospira genes such as, LipL32, Lig A and Lig B, and is effective for early diagnosis with clinical symptoms [35]. According to Harkin [36], the sensitivity and specificity to detect Leptospira genes from urine samples through PCR, can be as high as 100% and 88.3%, respectively. In contrast, the PCR assay has a 90% sensitivity in the first five days of illness, and greatly decreases to 50% if blood samples are used [37]. Since the dogs in this study all appeared healthy with no clinical signs, the negative PCR results were not unexpected. Urine samples should be used in the future study as it has higher sensitivity in comparison to the blood samples through PCR. Due to the widespread nature of this zoonotic disease, Leptospirosis is of public health concern. Therefore, it is important to determine the serovars that infect the dog population, as office handlers or care takers are constantly in contact with these animals. According to Masuzawa [38], the seroprevalence of L. australis and L. javanica among human beings in Phillipines was 8.5% and 1.4% (n=71), respectively. In addition, the seroprevalence of L. australis and L. javanica has also been detected among dairy farm workers in India, which was approximately 12.8% and 17.9% (n=41), respectively [39]. L. batavia was also reported causing tubulointerstitial renal failure in two children [40]. The seropositive results found in the current study, among the working dog population, suggests that humans are potentially at risk of
exposure, and the possibility of direct or indirect transmission of leptospirosis from dogs to human, cannot be ruled out. In addition to the routine annual vaccination, chemoprophylasis using doxycycline, might be useful in preventing and controlling canine leptospirosis among working dogs. The advantages of this preventive measure has been reported by the U.S. military [41]. Strict kennel sanitation, isolation of infected dogs and rodent control, are appropriate strategies to reduce the spread of leptospirosis. Routine herd screening of leptospirosis using MAT and PCR assay for working dogs, should be conducted to monitor the emergence of other serovars, and to provide early diagnosis for immediate and appropriate antimicrobial treatment.
Conclusion The seroprevalence of canine leptospirosis among the working dog population in Malaysia was 3.1% (n=3/96). The pathogenic infecting serovars detected include, L. australis, L. bataviae and L. javanica. To the best of our knowledge, this study is the first to report the seroprevalence of leptospirosis in working dogs from Malaysia. Effective preventative and control protocols for leptospirosis, should be implemented in order to maintain a healthy condition in both dogs and handlers.
Acknowledgements The authors would like to thank the staff from the Bacteriology Laboratory of the Faculty of Veterinary Medicine, Universiti Putra Malaysia for their assistance in processing the samples and technical help.
Table 1 List of the working dogs that showed antibody-antigen reaction to form agglutination during the microscopic agglutination test. Breed
Gender
Age (year)
Serovar
Antibody titer
Female
6
Javanica
1:160
Labrador
Male
9
Australis
1:80
German Shepherd
Male
4
Bataviae
1:80
German Shepherd
Female
6
Javanica
1:40
Labrador
Male
4
Icterohaemorrhagiae
1:40
Labrador
Male
7
Icterohaemorrhagiae
1:40
German Shepherd
Female
8
Lai
1:40
German Shepherd
Male
4
Pomona
1:20
Belgian Shepherd
Male
2
Hebdomadis
1:20
Labrador
Male
2
Javanica
1:20
German Shepherd
Male
3
Australis
1:20
German Shepherd
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