Performance of diagnostic assays used to detect Cryptosporidium oocysts in faecal samples of cattle in Kuwait and genotyping of Cryptosporidium species.

BACKGROUD: Cryptosporidium species are zoonotic protozoan parasites responsible for gastroenteritis in various animals and humans. The diagnosis of Cryptosporidium presents many challenges. This research attempted to match the diagnostic efficiency of the modified Ziehl-Neelsen technique (mZN), immunochromatographic assays (IC), and enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium in faecal samples of cattle in Kuwait. In addition, polymerase chain reaction (PCR) was utilised to determine the predominant species infecting cattle in Kuwait and correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle. RESULTS: Of 400 analysed faecal samples, Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN. IC had the highest sensitivity (74.07%), and mZN had the highest specificity (98.29%) using a composite reference standard (CRS) as a gold standard. The rapid IC test results in high false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. Only 74.5% (35/47) of Cryptosporidium-positive samples by the three assays could be amplified by PCR. This study was the first to genotype Cryptosporidium in Kuwait. Cryptosporidium parvum (n = 26) was the dominant species detected from cattle samples, followed by C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). The findings showed a statistically relevant relationship between diarrhoea and the detection of Cryptosporidium spp. in faecal samples of cattle (p-value = 0.0003). Pre-weaned calves were the most vulnerable age group to Cryptosporidium spp. infection (p-value = 0.0007). CONCLUSION: For screening of Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum was the prepoderant Cryptosporidium spp. recovered from cattle samples in Kuwait followed by C. andersoni. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.

Cross-sectional study and genotyping of rotavirus-A infections in ruminants in Kuwait.

BACKGROUND: Group A rotaviruses (RVA) are zoonotic pathogens responsible for acute enteritis in human and neonatal ruminants. This research aimed to determine the prevalence of RVA in ruminants (cattle, sheep, and goats) and investigate the circulating RVA genotypes in these animals in Kuwait. We conducted a cross-sectional study to detect RVA in ruminants, using an immunochromatography test (IC), direct sandwich ELISA test, and real-time RT-PCR (RT-qPCR) assay using fecal samples. RESULTS: A total of 400 cattle, 334 sheep, and 222 goats were examined. The prevalence of RVA was 5.3, 1.2, and 2.3%, respectively, using IC. The ELISA test detected RVA from 4.3% of cattle, 0.9% of sheep, and 1.8% of goats. There was a significant association between the occurrence of diarrhea and the presence of RVA in bovine fecal samples (p-value = 0.0022), while no statistical association between diarrhea and the presence of RVA in fecal samples of sheep and goats was observed (p-value = 0.7250; p-value = 0.4499, respectively). Twenty-three of the IC-positive samples (17 from cattle, two from sheep, and four from goats) were tested using a RT-qPCR RVA detection assay targeting the NSP3 gene. The results showed that 21 of 23 IC-positive samples tested positive by RT-qPCR. Detection of RVA genotypes revealed that G10P[11] was the predominant strain in cattle (58.8%), followed by G8P[1] (11.7%). One sheep sample was genotyped as G8P[1]. In addition, G6P[1] and G6P[14] were detected in goat samples. CONCLUSION: The present study revealed that the IC was more sensitive in detecting RVA antigen in fecal samples than the ELISA test. A higher occurrence of RVA infection was observed in cattle than in sheep and goats. This study suggests that RVA might be a risk factor of diarrhea in bovine calves less than 2 weeks old. This research also demonstrates the circulation of RVA in sheep and goat populations in Kuwait. Finally, the G10P[11] RVA genotype was the most prevalent genotype identified from cattle samples.

Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves.

Escherichia coli field isolates from calves were characterized and categorized into the most significant diarrheagenic pathotypes using polymerase chain reaction (PCR) assays with different specific primers. The used PCR systems were designed to detect sequences representing the group-specific virulence genes encoding fimbriae f5 (K99), Shiga toxins (stx1 and stx2), heat-stable enterotoxins (st), heat-labile enterotoxins (lt), intimin (eae), hemolysin (hylA), and EAEC heat-stable enterotoxin (astA). In the present work, a total of 150 E. coli field isolates were recovered from 150 fecal swabs collected from 100 diarrheic and 50 apparently healthy in-contact cattle and buffalo calves under 3 months old. Out of these 150 isolated E. coli, 106 isolates from 77 diarrheic and 29 in-contact calves harbored one or more of the investigated virulence genes. The pathotyping of the isolates could classify them into shigatoxigenic E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) with a 30.7, 2.7, 12.7, and 7.3% distribution, respectively. Meanwhile, the detection rates of f5, stx1, stx2, st, lt, eae, hylA, and astA genes were 17.3, 27.3, 6.7, 10, 37.3, 17.7, 9.3, and 20.7%, respectively. These virulence genes were found either single or in different combinations, such as stx/eae, stx/st/f5, eae/st/f5, or st/lt/f5. Four attaching-effacing shigatoxigenic E. coli isolates (AE-STEC) harboring stx/eae were retrieved from diarrheic calves. Although none of the stx-or eae-positive isolates was verified as O157:H7, STEC isolates detected in apparently healthy calves have potential pathogenicity to humans highlighting their zoonotic importance as reservoirs. Atypical combinations of ETEC/STEC and ETEC/EPEC were also detected in percentages of 14.7 and 2.7%, respectively. Most of these atypical combinations were found more in buffalo calves than in cattle calves. While STEC and EPEC isolates were detected more in cattle calves than in buffalo calves, ETEC isolates were the same in the two species. The pathogenic E. coli infection in calves was recorded to be higher in the first weeks of life with the largest numbers of virulence factor-positive isolates detected at the age of 4 weeks. Histopathological examination of five intestinal samples collected from four dead buffalo calves revealed typical attaching and effacing (AE) lesion which was correlated with the presence of intimin encoding virulence gene (eae). Other lesions characterized by hemorrhagic enteritis, shortening and fusion of intestinal villi and desquamation of the lining epithelium of intestinal mucosa had also been detected.

Epidemiological observations on cryptosporidiosis and molecular characterization of Cryptosporidium spp. in sheep and goats in Kuwait.

Molecular epidemiological analysis of cryptosporidiosis in Middle Eastern countries suggests that small ruminants could play a major role in the transmission of Cryptosporidium spp. to humans, with a dominance of Cryptosporidium parvum, especially its IId subtypes. However, little information is available on the epidemiology and risk factors of cryptosporidiosis as well the distribution of Cryptosporidium species/genotypes and subtypes in small ruminants in this area, including Kuwait. In the present study, 47 farms from several areas in Kuwait were visited once during October 2014 to September 2015 to collect data on risk factors associated with Cryptosporidium infection. Fecal samples from 334 sheep and 222 goats were examined for Cryptosporidium oocysts by Ziehl-Neelsen staining (ZN) and antigens by enzymatic immunoassay (EIA). The Cryptosporidium prevalence was higher when samples were examined by EIA than ZN (11.4 and 7.2% in sheep and goats by EIA, compared with 4.2 and 3.6% by ZN, respectively). Young age (less than 3 months) and closed housing system are risk factors of Cryptosporidium infection. A correlation between fecal consistency and the occurrence of Cryptosporidium spp. was observed; non-formed fecal samples were often found positive. Molecular characterization of 30 ovine and caprine samples using PCR-RFLP analysis of the small subunit rRNA gene revealed the presence of C. parvum in 23 samples, Cryptosporidium ubiquitum in five samples, and Cryptosporidium xiaoi in two samples. Sequence analysis of C. parvum at the 60 KDa glycoprotein gene locus identified two subtypes, IIaA15G2R1 and IIdA20G1, with the latter being more common (in 2 and 20 successfully subtyped samples, respectively). Only one subtype of C. ubiquitum (XIIa) was recorded. Cryptosporidiosis in small ruminants apparently poses public health problem in Kuwait.

Cross-sectional and histopathological studies of Feline Coronavirus infections in stray cats in Kuwait.

Feline Coronavirus (FCoV) is a worldwide viral infection of felids. The disease is usually asymptomatic, but it can cause mild diarrhoea; however, few numbers of cases may develop a severe systemic disease known as feline infectious peritonitis (FIP). This study aims to determine the prevalence of FCoV shedding in the faeces of stray cats in Kuwait and detect antibodies against FCoV in their serum. Histopathological analyses and RT­PCR were used to prove cases of FIP. A total of 178 cats were examined for the presence of FCoV in their faeces using a rapid immunochromatography (IC) test. Anti­FCoV Antibody (Anti­FCoV Ab) was detected in their serum using ELISA. Eleven samples were tested using RT­PCR to confirm positive cases. The prevalence of FCoV faecal antigen in stray cats was 32.6%. The overall detection rate of Anti­FCoV Ab in stray cats was 44.9%. Nine cats tested positive using the RT­PCR test. Six out of those nine were confirmed to be FIP positive through gross and histopathological examination. The characteristic uveitis and discoloration of the irises were seen. The present study is the first report confirming FCoV infection in stray cats in Kuwait. Postmortem and histopathological lesions in cases of FIP were recorded.

Epidemiological and Molecular Study of Cryptosporidium in Preweaned Calves in Kuwait.

Cryptosporidium is a worldwide enteric protozoan parasite that causes gastrointestinal infection in animals, including humans. The most notable species is Cryptosporidium parvum because of its zoonotic importance; it is also the leading cause of cryptosporidiosis in preweaned calves. A cross-sectional study was conducted to determine the prevalence of Cryptosporidium infection, investigate the potential risk factors, and use molecular diagnosis to identify the predominant Cryptosporidium spp. in preweaned calves in Kuwait. Of 175 preweaned calves, Cryptosporidium antigens were detected in 58 (33.1%) using rapid lateral immunochromatography assay (IC). Calves less than one month of age (OR = 4.32, p = 0.0001) and poor hygiene (OR = 2.85, p = 0.0075) were identified as significant risk factors associated with Cryptosporidium infection. Molecular identification revealed that C. parvum (62.8%) was the dominant species infecting preweaned calves in Kuwait. In contrast, C. bovis and C. andersoni were recorded at 5.7% and 2.9%, respectively. All C. parvum gp60 nucleotide sequences were subtype IIaA15G2R1. Calves could be a source of C. parvum infection due to the similarity of the subtypes recorded previously in Kuwaiti children and preweaned calves in this study. Therefore, more research is needed to understand the Cryptosporidium transmission cycle in Kuwait.

Evaluation of immunochromatography test for detection of four enteropathogens in the feces of sheep and goats in Kuwait.

Background: Diarrhea in newborn small ruminants continues to be the cause of significant financial loss in sheep and goat farms worldwide. Commercial immunochromatographic (IC) assays have been designed and evaluated to be used for the diagnosis of diarrhea in cattle; however, there are no trials to use rapid tests in small ruminants. Aim: This study was carried out in Kuwait to evaluate the performance of the rapid immunochromatography test (BoviD-4, BioNote, Inc, Korea) for diagnostics of Cryptosporidium, rotavirus A (RVA), bovine coronavirus (BCoV), and Escherichia coli K99 (E. coli K99) in fecal samples of sheep and goats. Methods: A total of 85 samples were examined using BoviD-4, and the results were compared with that of polymerase chain reaction for Cryptosporidium, RVA, and BCoV, whereas for E. coli K99 it was by isolation and identification as reference tests. Results: The kappa test agreement results between the BoviD-4 and reference tests were 0.870 (perfect), 0.783 (substantial), 0.728 (substantial), and 0.281 (fair) for the detection of E. coli K99, Cryptosporidium, RVA, and BCoV, respectively. The sensitivity of BoviD-4 kit was 91.2%, 80.0%, 90.0%, and 37.5% and the specificity was 88.2%, 96.0%, 96.4%, and 92.2% for Cryptosporidium, RVA, E. coli K99, and BCoV, respectively. Conclusion: The Bovid-4 kit can be used as a rapid pen-side test for Cryptosporidium spp., E. coli K99, and RVA in the field. Nonetheless, care must be taken while interpreting the BCoV results of the kit.

Increasing bedbug, Cimex lectularius, infestations in Kuwait.

Bedbug, Cimex lectularius, human infestations were reported in the State of Kuwait in the last 2 years. Eleven separate infestations from different localities were received at the Veterinary Laboratories indicating that bedbug is widespread in the State of Kuwait. There was circumstantial evidence to suggest the transfer of bugs with recent immigrants or used furniture. The spread of infestation can be attributed to the increase in migrant labor and their mobility inside the country. The increase in reported cases appears also consistent with a worldwide increase in bedbug infestations.

Enteric protozoan parasites in stray cats in Kuwait with special references to toxoplasmosis and risk factors affecting its occurrence.

In Kuwait, stray cats were surveyed for enteric protozoan infection using fecal examination and their sera were tested for Toxoplasma gondii IgG using indirect hemagglutination test (IHAT) as well as for feline immunodeficiency virus (FIV) antibodies and feline leukaemia virus (FeLV) antibodies using ELISA. Out of 240 fecal samples examined 22 (9.2%) were found to be infected with oocysts of four species of coccidian protozoa. Isopspora felis was the most predominant enteric protozoan parasite (7.1%), followed by T. gondii (2.1%), I. rivolta (1.6), Sarcocystis was only found in one case (0.4%). Juvenile cats ( 6 months old) had higher infection rate with oocyst of enteric protozoa than older cats (p-value 0.001). Sero-survey of 240 stray cats revealed that 19.6% were positive to T. gondii IgG. Toxoplasma sero-positivity was observed in higher number of adults compared to young cats suggests that with age the risk of exposure to T. gondii increases. While concurrent retroviral infections were not found to be associated with increased risk for developing T. gondii antibodies.