High Asymptomatic Cases of Babesiosis in Dogs and Comparison of Diagnostic Performance of Conventional PCR vs Blood Smears.

PURPOSE: Babesiosis is one of the most important globally extended and quickly spreading tick-borne infections of dogs. Diagnosis of babesiosis in Sri Lanka is based on clinical signs followed by thin blood smears which could be error-prone due to undetected early infections, absence of clinical signs or low parasitemia. The present study investigated the prevalence of babesiosis in dogs presented to the Veterinary Teaching Hospital (VTH) at the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, for treatments, vaccinations, and regular check-ups, and compared the diagnosis methods of microscopy and molecular analysis. METHODS: Blood samples from dogs were collected from January to June 2019. First, Giemsa stained blood smears were prepared, and then the blood samples were subjected to PCR using genus-specific primers to amplify a 411-450 bp region in the 18S rRNA gene. Twenty samples from PCR amplified products were sequenced for species identification and phylogenetic analysis. Clinical signs of the dogs were noted down, and ticks were also collected from dogs if any. RESULTS: Results show a very high prevalence of canine babesiosis (78.6%) among the dogs brought to the VTH. The parasite was identified microscopically and genetically as Babesia gibsoni. A large percentage (66.7%) of infections was asymptomatic. Out of 42 blood samples, 19 (45.2%) were microscopically positive for babesiosis while 33 (78.6%) were PCR positive, showing a significant difference in the two methods of diagnosis (chi-square test, χ2 = 9.462, p = 0.002). Three tick species: Rhipicephalus haemaphysaloides, Haemaphysalis bispinosa, and Rhipicephalus sanguineus were found attached to the dogs. CONCLUSION: This study shows a very high prevalence of canine babesiosis among dogs in the Kandy area. Most of these infections might go undetected if only microscopy was used to diagnose. An improved, rapid diagnostic method such as the novel, PCR-based point-of-care diagnostic method that detects very low parasitemia within 30 min is needed. Moreover, as most infected dogs did not show clinical signs, they may act as reservoirs of infection. The ability of asymptomatic dogs to spread babesiosis should be investigated.

Canine trypanosomosis in Sri Lanka: An emerging problem reported from three distinct geographic locations.

Here we report three cases of canine trypanosomosis presented to the Veterinary Teaching Hospital in the Faculty of Veterinary Medicine and Animal Sciences at the University of Peradeniya, Sri Lanka during 2018. The cases were presented to the hospital when the dogs were already in critical condition. Confirmation of the cases was done by microscopic examination of Giemsa-stained thin blood smears. All three dogs had bilateral keratitis and anterior chamber cloudiness in eyes. Despite the intramuscular administration of diminazine aceturate, all of them subsequently died. Amplification and sequencing of a fragment of the internal transcribed spacer 1 (ITS1) of the nuclear ribosomal DNA confirmed the parasite as Trypanosoma. evansi. This is the first record of clinical cases of canine trypanosomosis in Sri Lanka. The three cases reported here came from widely separated geographical locations within the country: Balangoda, Mullaitivu and Kadawatha.

Seroprevalence of Toxoplasma gondii antibodies in captive elephants (Elephaus maximus maximus) in Sri Lanka.

Serum samples collected during August 2003-June 2004 from 45 privately owned captive and 8 elephants from the Pinnawala Elephant Orphanage were tested for the presence of antibodies against Toxoplasma gondii using the direct modified agglutination test (MAT). Antibodies were found in sera of 14 of 45 (32%) privately owned elephants with titers of 1:25 in three, 1:50 in three, 1:100 in three, 1:200 in three, and 1:400 in three elephants. The elephants from Pinnawala Elephant Orphanage were seronegative. This is the first report of T. gondii seroprevalence in elephants in Sri Lanka.

Reliable noninvasive genotyping: fantasy or reality?

Noninvasive genotyping has not gained wide application, due to the notion that it is unreliable, and also because remedial measures are time consuming and expensive. Of the wide variety of noninvasive DNA sources, dung is the most universal and most widely used in studies. We have developed collection, extraction, and amplification protocols that are inexpensive and provide a high level of success in amplifying both mitochondrial and nuclear DNA from dung. Here we demonstrate the reliability of genotyping from elephant dung using these protocols by comparing results from dung-extracted DNA to results from blood-extracted DNA. The level of error from dung extractions was only slightly higher than from blood extractions, and conducting two extractions from each sample and a single amplification from each extraction was sufficient to eliminate error. Di-, tri-, and tetranucleotide loci were equally reliable, and low DNA quantity and quality and PCR inhibitors were not a major problem in genotyping from dung. We discuss the possible causes of error in genotyping with particular reference to noninvasive samples and suggest methods of reducing such error.

Seroepidemiology of Brucella abortus infection in bovids in Sri Lanka.

From 1992 to 1995, 0.15% (n=3916) of the bovids (cattle and buffalo) in Sri Lanka were sampled, using a multi-stage sampling procedure. Serum antibodies for Brucella abortus were detected using the indirect enzyme-linked immunosorbent assay (ELISA). The age, the agroecological zone and the management system practiced in the farms of the sampled bovids were studied as risk factors for seropositivity. The overall seroprevalence of brucellosis in cattle was 4.7% (n=3076) and 4.2% in buffaloes (n=840). Bovids that were over 3 years of age, from the dry zone (annual rainfall 20-35 in.), and reared under an extensive management system had higher odds of being seropositive. Bovids from the dry zone were at approximately six times higher odds of being seropositive even after controlling for the possible effects of age and management system. Approximately 75% of the seropositive males were from the dry zone. Most bovids (84%) from the dry zone in this study were reared under an extensive management system (free grazing) which allows unrestricted contact between animals. These factors may have contributed to the spread of brucellosis in bovids in the dry zone. This infection might be an important cause of abortion in bovids in Sri Lanka. However, there is also evidence of other causes for abortion, repeat breeding and retained placenta.

The relationship between faecal egg count reduction and the lethal dose 50% in the egg hatch assay and larval development assay.

The relationship between resistance detected in the faecal egg count reduction test (FECRT) and the lethal dose 50% (LD50) in the egg hatch assay (EHA) for benzimidazoles (BZs) and a larval development assay (LDA) for BZs, levamisole (LEV) and ivermectin (IVM) was examined on 13 sheep farms and 12 goat farms in Denmark. Out of 10 farms where resistance to BZs was detected according to the FECRT, nine (90%) had LD50 values above 0.5 microM thiabendazole (TBZ) (0.1 microg TBZ/ml) in the EHA, indicating resistance to BZs. However, four out of the 12 isolates susceptible to BZs in the FECRT had LD50 values higher than 0.5 microM TBZ in the EHA. For all isolates examined, LD50 values for TBZ in the LDA were lower than in the EHA. Four out of 11 and five out of 12 farms with worm populations resistant to BZs according to the FECRT and EHA respectively, had LD50 values lower than 0.5 microM TBZ in the LDA. Using the same cut-off point for resistant isolates in the LDA as in the EHA (0.5 microM TBZ), these isolates would be considered susceptible to BZs. All 10 isolates susceptible to BZs according to the FECRT and EHA and two isolates with suspect BZ resistance had LD50 values lower than 0.5 microM TBZ in the LDA. The above results indicated fairly good agreement in the detection of BZ resistance between the FECRT, EHA and the LDA. Groups of farms where resistance to LEV was detected according to the FECRT had higher mean LD50 values compared to those with LEV-susceptible or suspected resistant isolates. However, only four out of 12 farms having isolates resistant to LEV had LD50 values higher than 1.2 microM LEV (0.28 microg LEV/ml) recorded previously for a LEV-susceptible strain of Ostertagia circumcincta. This indicated discrepancies in declaring resistance to LEV between the FECRT and the LDA. Isolates from four farms where resistance to IVM was detected in the FECRT had LD50 values higher than the susceptible isolates. These were 2.5 to 7.5 times higher than those recorded previously for IVM-susceptible strains.

Seroepidemiology of rinderpest in bovids in Sri Lanka using the enzyme linked immunosorbent assay (ELISA) technique.

Approximately 0.2% (n = 4397) of the bovids (cattle and buffalo) in Sri Lanka were sampled, from June 1992 using a multi-stage sampling procedure. Serum antibodies for the rinderpest virus were detected using the competitive enzyme-linked immunosorbent assay. The age, the agroclimatic zone, the management system practiced in the farms, and the vaccination history of the sampled bovids were studied as potential risk factors for being seropositive. The prevalence of rinderpest antibodies in non-vaccinated bovids was 3.5% (n = 4101). The prevalence was higher in the dry zone (9%; where the outbreak emerged in 1987), compared to bovids in the other zones (1%). Seropositive bovids over three years of age were approximately at fourfold higher chances of being seropositive compared to those that were < or = 3 years old. The higher prevalence in older animals is probably due to exposure to the virus during the 1987 epidemic. Bovids from the dry zone (annual rainfall 20 to 35 inches) were at higher odds of being seropositive even after controlling for the possible effects of age, agroclimatic zone, management system and vaccination. The fact that 62% of bovids from the dry zone in this study were reared under extensive management system (free grazing) which allow unrestricted contact between animals, may be the reason for the above finding. A relatively poor response to vaccination observed in vaccinated bovids (seroprevalence = 12%; n = 296) could be attributed to difficulties in maintaining the vaccine at recommended temperatures in the field. This is the first island-wide study on seroprevalence of rinderpest in Sri Lanka.

Worm control practices on sheep farms in Nyandarua District of Kenya.

A questionnaire investigation was used to examine anthelmintic usage and practical worm control for sheep on 50 farms selected randomly in Nyandarua District of Central Kenya. Control of helminths was based primarily on the use of anthelmintics on all 50 farms. On the majority (54%) of these properties, lambs were drenched two times per year. Ewes and rams were drenched three or four times per year on 74% of the farms. Most treatments were given at intervals of approximately 3 months with no specific drenching programmes. Anthelmintic doses for the sheep were based on weights estimated using visual appraisal on 98 and 96% of the properties for lambs and adult sheep, respectively. Only on a small proportion of the farms (22%) was the recommended weight of the heaviest animal used when drenching groups of either lambs or adult sheep. In 1994, the majority (68%) of farmers used levamisole (LEV) in combination with oxyclosanide (OXY) a fasciolicide, 10% used benzimidazoles (BZs), 10% LEV alone and 12% LEV and BZs together. This pattern of anthelmintics use was maintained from 1988 to 1994. Eighty one percent of the farmers had been using only LEV or BZs for three or more consecutive years from 1990 to 1994. The implications of these findings for the development of anthelmintic resistance are discussed.

Faecal egg count reduction percentage calculations to detect anthelmintic resistance in Oesophagostomum spp. in pigs.

The results of four alternative methods of mean faecal egg count reduction percentage (FECR%) calculations were evaluated and compared using data obtained for Oesophagostomum spp. from ten sow herds. The estimates of FECR% and 95% confidence limits obtained using the four methods were different. However, there were few discrepancies in the final decision as to whether a given herd carried drug resistant isolates or not. The methods that used geometric means were more appropriate than those that used the arithmetic mean as the measure of central tendency for eggs per gram of faeces (EPG) values. The use of geometric mean EPG values in calculations has been criticized from several viewpoints, one of which is that its use reduces the comparability of reports between laboratories. If the geometric mean is to be used as we suggest in FECR% calculations, the appropriate references, number of animals in each group, minimum and maximum EPG values and the factor added to zero EPG counts should be reported in order to improve the comparability. The difficulty in obtaining groups with similar pre-treatment EPG values in field situations suggested the inclusion of pre-treatment EPG values in the calculations as an adjustment procedure. The importance of including a non-treated control group in calculations was demonstrated during this study. Therefore, we suggest the use of geometric mean EPG values, to include pre-treatment EPG values and to include the egg counts from the control group in FECR% calculations. The interpretation of the resulting FECR estimate may be different according to the purpose for which the testing procedure is carried out, e.g. survey in detecting anthelmintic resistance, control field tests, etc.

A questionnaire survey of nematode parasite control practices on goat farms in Denmark.

A questionnaire survey to examine worm control practices on angora and other goat farms in Denmark was undertaken in 1993. Questionnaires were inserted in the July, 1993 issue of the official journal of the angora goat farmers in Denmark (Mohair News) and 72 questionnaires posted directly to other goat farms selected from a list of 150 farms registered with the Goat Breeders Association. Data from 51 angora and 41 other goat farms indicated that anthelmintics were used on more than 80% of the farms. On 51% of the farms, no predetermined drenching programmes were followed. On 72% and 64% of the farms, kids (< 1 year of age) and adult goats (> 1 year old) were drenched 1-3, and two or three times per year, respectively. Only 9% of the farmers used the recommended weight of the heaviest animal in a herd to determine anthelmintic doses for groups of both kids and adult goats. Visual perception which may result in underdosing was used to determine weights for drenching on 41% and 69% of the farms for kids and adult goats, respectively. From 1986 to 1993, Class I anthelmintics (benzimidazoles) were the most commonly used. Anthelmintic classes were not alternated annually on the majority of farms, which would enhance selection for resistance. While the majority of farmers (58%) rotated goats regularly between paddocks, only 21% drenched the goats when being moved. Anthelmintic treatment for newly introduced goats was not carried out on 45% of 55 respondent farms, nine of which had imported stock. This increases the likelihood of introducing resistant worms to the farms.

Worm control practices on sheep farms in Denmark and implications for the development of anthelmintic resistance.

A questionnaire survey was undertaken on sheep farms in Denmark to obtain information on sheep management and worm control practices. The questionnaires were inserted in the June and July, 1993 issue of the Danish Sheep Breeders Journal, circulated monthly to approximately 1200 sheep breeders nationwide. A total of 183 (11%) questionnaires were returned. A second questionnaire was posted to 50 of the 183 responding farmers to examine the repeatability of responses to the first questionnaire. The majority (97%) of the 183 farmers used anthelmintics. The mean number of doses per year for lambs (< 12 months old) and adult sheep (> 12 months old) were 1.9 and 2.3, respectively. Only 42% of the farms followed predetermined drenching programmes. Live weights were determined by visual appraisal on 45% and 84% of the farms for lambs and adult sheep, respectively. In calculating anthelmintic doses for groups of lambs and adult sheep, the recommended weights of the heaviest animals were only used on only 18% and 27% of the farms respectively. Benzimidazoles were the most commonly used anthelmintics from 1986 to 1993. Eighty one percent of 94 responding farmers had been using the same class of anthelmintic for 3 or more consecutive years. Information from this study indicated lack of knowledge about worm control strategies, anthelmintic use and the problem of anthelmintic resistance among the majority of Danish sheep farmers. The majority of responses to the questionnaire were repeatable at least to a fair degree except when farmers were asked to recall anthelmintics used in past years.

A questionnaire investigation on factors of importance for the development of anthelmintic resistance of nematodes in sow herds in Denmark.

A questionnaire survey on the use of anthelmintics and presence of other potential risk factors for the development of anthelmintic resistance of nematodes in 136 Danish sow herds was carried out between November 1992-February 1993. Twenty one veterinarians who specialize in diseases of pigs administered the questionnaire in personal interviews during their regular visits to the respective pig farmers. In the study population, 91% of the farmers currently treated their sows with anthelmintics, while only 38% and 14% treated their weaners and fatteners, respectively. Most respondents treated their sows either before or after farrowing (40%) or once every 6 months (27%). Benzimidazole and pro-benzimidazole (Class I) anthelmintics were the most used in sows by the farmers (39%, 47%, 49%, 50%, 46%, and 47% during the years 1987, 1988, 1989, 1990, 1991 and 1992, respectively). When sows were treated, fenbendazole was the most frequently used anthelmintic during the period 1987-1992 (30%-39%). The proportion of farmers that used Class II (pyrantel and levamisole) or Class III (ivermectin and piperazine) anthelmintics during the year 1992 were 25% and 13%, respectively. Of the 70 farmers who used anthelmintics from either Class I, II or III during the study period, 44 farmers always used the same class of drugs. Eight herd owners among those 44, had changed the pharmaceutical product within the same class. Sixty nine percent of the farmers did not weight the sows, but used visual appraisal of average body.

A study on the transmission of Oesophagostomum dentatum and Hyostrongylus rubidus among outdoor reared pigs in Denmark.

This study was carried out to obtain basic information on the transmission of Oesophagostomum dentatum and Hyostrongylus rubidus in outdoor reared pigs in Denmark. Eighteen 10 weeks old worm-free pigs were allocated into 3 groups of 6 pigs each. In May, all pigs were turned out on the same parasitologically naive pasture, and after 2 weeks the pigs in groups 2 and 3 were experimentally infected with 10,800 O. dentatum and 8,700 H. rubidus infective larvae, respectively. Pigs in group 1 served as non-infected controls. All pigs were reared together on the experimental pasture for further 134 days until slaughter in October. Strongyle egg counts, differentiation of infective larvae at species level, serum pepsinogen, and herbage larval infectivity were monitored at regular intervals throughout. Both strongyle species established in the originally parasite-free pigs (group 1) and cross infections were established in group 2 and 3. The pigs were exposed to steadily increasing herbage infectivity of both species of strongyles. At the end of the experiment, geometric mean worm burdens of O. dentatum in groups 1, 2 and 3 were 1202, 6136 and 1431 respectively, the burden in group 2 being significantly higher (p < 0.05) than that of the 2 other groups. The geometric mean worm burdens of H. rubidus in groups 1, 2 and 3 were 4907, 3679 and 5246 respectively, showing no significant differences between groups.