Diagnostic Analysis of mZN Microscopy and PCR for Detection of Cryptosporidiosis in Goat Kids - A Molecular Epidemiological Study

The occurrence of Cryptosporidium species infection and its risk factors in neonatal goats is less explored. Also it is due to the fact that diseases like colibacillosis and neonatal viral enteritis complex caused by Group A rotaviruses and Bovine corona viruses can co-exist with Cryptosporidium and can lead to mixed infections and the latter is often overlooked. Therefore, in the current research we explored the cryptosporidial occurrence in neonatal goats of Mathura district of Uttar Pradesh, India. In this study, a total of 644 faecal samples were collected from neonatal goats at different villages and certain organized farms of Mathura district age-wise, season-wise and breed-wise, and were examined for Cryptosporidium based on modified Ziehl-Neelsen technique, conventional 18SSU rRNA nested PCR assay. The overall prevalence of Cryptosporidium infection in goats based on microscopy was 36.80% (237/644;p value <0.0001) and 18SSU rRNA nested PCR 52.95% (341/644;p value <0.0001) respectively. Cryptosporidium species typing was also done using 18SSU rRNA nested PCR-RFLP product using enzymes Mbo-II, Ssp-I and Vsp-I, which revealed species including C. parvum C. bovis, C. ryanae, C. hominis and C. andersoni. Also the infection was clinically associated based on age, gender and seasons to identify the causal relationships that precipitate the cryptosporidial infection in goat kids. Since mZN microscopy based screening requires expertise and may sometimes be confuse with other weak acid fast bodies and also due to low sensitivity, combination of diagnostic tests are used in this study to identify the best test combination that yields best statistical fit in terms of kappa-agreement and McNemar's test. Cryptosporidiosis is caused by an enteric protozoan parasite and the first report in sheep and goat was observed in early 1980s, with other important etiological agents for neonatal diarrhoea, mortality and morbidity in neonatal kids and lambs, responsible for economic losses.Copyright © 2023 Indian Veterinary Assocaition. All rights reserved.

An end-point multiplex PCR/reverse transcription-PCR for detection of five agents of bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is frequently associated with single or mixed viral, bacterial and/or protozoal infections. Consequently, laboratory diagnostic of NCD usually requires specific tests for each potential agent; a time-consuming, laborious and expensive process. Herein, we describe an end-point multiplex PCR/reverse transcription-PCR (RT-PCR) for detection of five major NCD agents: bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (E. coli K99), Salmonella enterica (S. enterica) and Cryptosporidium parvum (C. parvum). Initially, we selected and/or designed high-coverage primers. Subsequently, we optimized multiplex PCR/RT-PCR conditions. Next, we evaluated the analytical sensitivity of the assay and assessed the performance of the reaction by testing 95 samples of diarrheic calf feces. The analytical specificity was evaluated against bovine viral diarrhea virus (BVDV), E. coli heat-stable enterotoxin (STa) and Eimeria spp. The detection limit of our assay was about 10 infectious units of BRV, 10-2 dilution of a BCoV positive sample pool, about 5 × 10-4 CFU for S. enterica, 5 × 10-6 CFU for E. coli K99 and 50 oocysts for C. parvum. No non-specific amplification of other bovine diarrhea agents was detected. Out of 95 samples analyzed, 50 were positive for at least one target, being 35 single and 15 mixed infections. BRV was the most frequent agent detected in single infections (16/35), followed by Cryptosporidium spp. (11/35), which was the most frequent in mixed infections (11/15). Positive and negative multiplex results were confirmed in individual reactions. In conclusion, we described an end-point multiplex PCR/RT-PCR for faster and easier NCD diagnosis, which may be useful for routine diagnosis and surveillance studies.

Viral enteritis in cattle: To well known viruses and beyond

Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Pathogen-oriented approaches for neonatal calf diarrhea

Neonatal calf diarrhea (NCD) is the leading health concern in calves during the first weeks of their lives. In this narrative review, the potential for pathogen-oriented approaches for NCD is discussed. The literature on NCD clearly shows substantial differences in spread and characteristics between the major NCD pathogens, making pathogen-oriented approaches possible, justifying the use of etiological diagnostics. For enterotoxic Escherichia coli, colostrum delivery and dam vaccination, biosecurity around calving and antimicrobial therapy are key. Both for bovine coronavirus (BCV) and bovine rotavirus (BRV), biosecurity and disinfection, dam vaccination in combination with adequate and prolonged colostrum delivery are the essentials. However, a different focus concerning biosecurity is necessary given the airborne spread of BCV and higher environmental persistence of BRV. For an effective Cryptosporidium spp. control, the use of disinfectants that kill oocysts is crucial. Evidence supporting the prophylactic use of halofuginone lactate to reduce shedding and diarrhea, is available, but in terms of biosecurity, attention should be placed on the proper use of this product. In case of a Salmonella enterica outbreak, antimicrobial use remains important, and biosecurity wise, attention should be paid to shedding of periparturient cows in the calving pen and administration of infected colostrum. Both for S. enterica and cryptosporidiosis, farm staff should be informed on how to protect themselves against these zoonotic infections. Nutritional factors play an additional role within NCD. Improper nutrition management can induce diarrhea or can further enhance infectious NCD through osmosis or dysbiosis. In conclusion, the suggested pathogen-oriented approaches can aid to economize labor and financial investments, limit the environmental impact of NCD control and prevention and valorize tailor-made farm advisory work.

Evaluation of immunochromatographic point-of-care tests for the detection of calf diarrhoea pathogens in faecal samples

We have evaluated the diagnostic performance of immunochromatographic point-of-care tests (POCT) for the detection of rotavirus, coronavirus, Escherichia (E.) coli F5, Cryptosporidium (C.) parvum, Clostridium (Cl.) perfringens and Giardia (G.) intestinalis in fresh and thawed faecal samples from calves aged up to six months with diarrhoea. We performed POCTs to detect rotavirus, coronavirus, E. coli F5, C. parvum, Cl. perfringens and G. intestinalis on fresh samples in a field study and re-evaluated the performance for C. parvum, Cl. perfringens and G. intestinalis using thawed samples. We calculated the performance based on the results of the reference methods, which were RT-qPCR for the detection of rota- and coronavirus and bacteriological culturing and PCR to detect E. coli F5 and Cl. perfringens a and ss2 toxins. C. parvum was detected by phase-contrast microscopy and G. intestinalis by immunofluorescence microscopy. We collected 177 faecal samples from diarrhoeic calves. We found good performance for the POCT targeting rotavirus (sensitivity (SE)=92.9%;specificity (SP)=95.6%) and C. parvum (SE=63.3%;SP=96.2%). For E. coli F5, the number of true positive samples (n=1) was too low to evaluate the performance. The POCT to detect coronavirus gave a poor performance (SE=3.3%;SP=96.6%) and the POCT to detect Cl. perfringens a moderate performance (SE=52.8%;SP=78.2%). G. intestinalis POCT showed a higher sensitivity to immunofluorescence microscopy in thawed than in fresh faecal samples (SE=43.9% versus SE=29.2%). There are substantial differences in diagnostic performance between the commercially available immunochromatographic POCTs. Still, POCT can make a valuable contribution to the diagnosis and prevention of calf diarrhoea.

Enteritis in calves: anamnestic, clinical, diagnostic and therapeutic evaluations with special attention to enteropathogenic rotavirus and bovine coronavirus

Introduction: Neonatal calf diarrhoea is a multifactorial disease that sometimes leads to high economic losses. It can be fatal due to dehydration and acidosis and has been one of the main causes of calf mortality. Material and methods: This retrospective study considered calves of a maximum of 35 days of age and with a diagnosed infection with rotavirus and/or bovine coronavirus. We examined the clinical records of 156 calves that were referred to the University Clinic for Ruminants in Vienna. Results Calves that had been treated with antibiotics before admission to the Clinic had a higher risk of staying longer, suggesting either that these calves had a more serious illness or that antibiotic treatment was not indicated and so therapeutic success was not achieved. Twenty-three calves died or were euthanized at the Clinic. At the time of admission, they were younger than the surviving calves and they had a lower inner body temperature and a lower base excess at the first examination. The four most common pathogens in faecal samples were rotavirus, bovine coronavirus, Cryptosporidium parvum and Escherichia coli, which were detected in 67.1%, 53.9%, 48.1% and 94.1% of the faecal samples examined. The most common co-infection was rotavirus with Cryptosporidium parvum (17 faecal samples). We inspected the four most common pathogens in more detail. There were significant correlations between bovine coronavirus and season, with the risk of suffering from bovine coronavirus 1.6 times higher in winter than in other seasons. There was also a correlation between Cryptosporidium parvum and general behaviour: the risk of being infected with Cryptosporidium parvum was 2.6 times higher in calves that were moderately to severely depressed at the first examination. There was a correlation between co-infections and mortality, with calves with a co-infection at three times higher risk of dying than calves with a mono-infection.

Development and Application of an Insulated Isothermal RT-PCR for On-site Detecting Nebovirus

Nebovirus (NeV) is an emerge diarrhea-causing virus in calves, the aim of this study is to establish an insulated isothermal RT-PCR(iiRT-PCR) for detecting NeV on field. Based on the RdRp sequences of NeV in GenBank database, a pair of primers and a fluorescent TaqMan probe were designed and synthesized. After optimizing the react system and condition, the iiRT-PCR method for detection of NeV was established. The iiRT-PCR assay could amplify specific fragment of NeV, without amplification of irrelevant pathogens, including bovine coronavirus, bovine norovirus, bovine rotavirus, bovine viral diarrhea virus, bovine torovirus, bovine Cryptosporidium parvum, bovine Eimeria. The intra- and inter-coefficients of variation were 3.07%-3.12% and 2.45%-3.01%, respectively, and the detection limit of viral nucleic acid of the assay was 5.38 copies•μL-1. One hundred and one calf diarrhea samples, collected from Hongyuan County, Ruoergai Prefecture, Xichang City, Liangshan Yi Autonomous Prefecture and Langzhong City in Sichuan Province during 2020-2021, were used to detect NeV, and 64.36% samples were detected as NeV positive. The study established an iiRT-PCR method for NeV detection with good specificity and reproducible as well as high sensitivity. Moreover, combined with the premixed detection reagent and PetNAD nucleic acid extraction kit, this assay could be used to NeV detect on-site, and only 1 hour from nucleic acid extraction to result report, which contribute to the fast detection for NeV.

Diarrhoea outbreak caused by coinfections of Cryptosporidium parvum subtype IIdA20G1 and rotavirus in pre-weaned dairy calves.

Diarrhoea is one of the most important syndromes in neonatal calves. In industrialized nations with intensive animal farming, Cryptosporidium spp. and rotavirus are primary causes of calf diarrhoea, but the role of these and other enteric pathogens is not clear in China. In November and December 2018, a diarrhoea outbreak was identified in over 150 pre-weaned calves on a dairy farm in Heilongjiang Province, northeast China and approximately 60 calves died. To determine the cause of the outbreak, we analyzed 131 faecal samples collected from pre-weaned calves (0-2 months) during (n = 114) and after the outbreak (n = 17). Initially, 10 diarrheic samples during the outbreak and 10 non-diarrheic samples after the outbreak were screened for rotavirus, coronavirus, Escherichia coli K99 and Cryptosporidium parvum by using an enzymatic immunoassay (EIA). In addition, 81 other samples were tested specifically for rotavirus by EIA, and all 131 samples were analyzed for Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi by PCR. The initial EIA analysis identified C. parvum (8/10) and rotavirus (5/10) as the dominant pathogens in calves during the outbreak, while both pathogens were detected at lower frequency after the outbreak (2/10 and 1/10, respectively). Further PCR analyses indicated that the occurrence of C. parvum infections in calves was significantly higher during the outbreak (75.4%, 86/114) than after the outbreak (11.8%, 2/17; odds ratio [OR] = 23.0), and was significantly associated with the occurrence of watery diarrhoea (OR = 15.7) and high oocyst shedding intensity. All C. parvum isolates were identified as subtype IIdA20G1. Among other pathogens analyzed, the overall prevalence of rotavirus, G. duodenalis and E. bieneusi was 19.8% (20/101), 38.9% (51/131) and 42.0% (55/131) in calves, respectively, without significant differences during and after the outbreak. Among the three pathogens, only the rotavirus infection was associated with diarrhoea in calves. More importantly, coinfections of C. parvum and rotavirus were significantly associated with the occurrence of watery diarrhoea in calves and were seen only during the outbreak. Thus, C. parvum subtype IIdA20G1 and rotavirus appeared to be responsible for this diarrhoea outbreak. Control measures should be implemented to effectively prevent the concurrent transmission of these enteric pathogens in pre-weaned dairy calves in China.

Occurrence and genetic diversity of rotavirus A in faeces of diarrheic calves submitted to a veterinary laboratory in Spain.

A total of 237 faecal specimens from diarrheic calves younger than two months were collected and submitted for diagnosis of enteropathogens over a two-year period (2017-2018) to a veterinary laboratory. Samples originated from 193 dairy and beef farms in 29 provinces distributed throughout Spain, and were tested for the occurrence of three target enteric pathogens by reverse transcription real-time PCR (RT-qPCR): bovine rotavirus A (RVA), Cryptosporidium parvum and bovine coronavirus (BCoV). RT-PCR and nucleotide sequencing analysis were used to determine the G (VP7 gene) and P (VP4 gene) genotypes of 26 specimens positive for RVA. A total of 188 specimens (79.3 %) were positive for at least one of the three target enteric pathogens, and 101 samples (42.6 %) harbored mixed infections. The individual prevalence was 57.8 %, 50.6 % and 23.6 % for C. parvum, RVA and BCoV, respectively. Molecular analysis of selected RVA strains revealed the presence of the G6, G10, G3, P[5] and P[11] genotypes, with the combinations G6P[5] and G6P[11] being the most prevalent. Alignments of nucleotide sequences of the VP7 and VP4 markers showed a high frequency of single nucleotide polymorphisms (SNPs), with up to 294 SNPs found in 869bp of sequence at the G6 genotype (0.338 SNPs/nt), which reveals the extensive genetic diversity of RVA strains. Phylogenetic analysis of the VP7 gene of the G6 strains revealed four distinct lineages, with most strains clustering in the G6-IV lineage. The discrepancies between the RVA genotypes circulating in the sampled cattle farms and the genotypes contained in commercial vaccines currently available in Spain are discussed. We believe that this is the first study on the molecular characterization of rotavirus infecting cattle in Spain.

First report on Cryptosporidium parvum, Escherichia coli K99, rotavirus and coronavirus in neonatal lambs from north-center region, Algeria.

The etiology of neonatal diarrhea is multifactorial and remains one of the greatest health problems in sheep livestock farming. Faecal samples from 559 neonatal lambs aged less than 30 days from 30 sheepfolds located in the north-center region of Algeria were screened with pathogen-specific antigen ELISA for Cryptosporidium parvum, Escherichia coli K99, rotavirus, and coronavirus. Of the 559 lambs, 312 (58.81 %), 155 (27.72 %), 72 (12.88 %) and 20 (3.57 %) were positives for C. parvum, E. coli K99, rotavirus and coronavirus antigens, respectively. The prevalence of C. parvum was the highest (p < 0.0001). C. parvum, E. coli K99, rotavirus and coronavirus were observed in 23 (76.66 %), 17 (56.66 %), 9 (30 %) and 3 (10 %) sheepfolds, respectively. Compared to age, the prevalence of C. parvum was highest during the second and third week of age (p < 0.001). In contrast, other pathogens were found to be more frequent in lambs aged ≤7 days (p < 0.001). The number of lambs with diarrhea was 280 (50.09 %) of which 280 (100 %), 127 (45.35 %), 52 (18.57 %) and 10 (3.57 %) were found to be infected with C. parvum, E. coli K99, rotavirus and coronavirus, respectively (p < 0.0001). In various combinations, mixed infections were detected only with C. parvum. This is the first report of C. parvum, E. coli K99, rotavirus, and coronavirus in ≤30-days old neonatal lambs in Algeria. Special attention should be given to the first colostrum feeding, hygiene of the farm, prevention and control measures for a better prevention of neonatal diarrhea in lambs.