Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea

Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide. Data on the prevalence of Cryptosporidium spp. and its subtypes among calves in the Republic of Korea (KOR) are sparse. Hence, our study aimed to investigate the prevalence and association between the age of calf and the identified Cryptosporidium spp. and to determine the genotypes/subtypes of Cryptosporidium spp. in pre-weaned calves with diarrhea in the KOR. A total of 460 diarrheic fecal samples were collected from calves aged 1−60 days and screened for Cryptosporidium spp. by the 18S rRNA gene. Species identification was determined using the sequencing analysis of the 18S rRNA gene, and C. parvum-positive samples were subtyped via the sequence analysis of the 60-kDa glycoprotein (gp60) gene. Sequence analysis based on the 18S rRNA gene revealed the presence of three Cryptosporidium spp., namely, C. parvum (n = 72), C. ryanae (n = 12), and C. bovis (n = 2). Co-infection by these species was not observed. The infection rate was the highest in calves aged 11−20 days (26.1%, 95% CI 17.1−35.1), whereas the lowest rate was observed in calves aged 21−30 days (7.7%, 95% CI 0.0−16.1). The prevalence of C. parvum was detected exclusively in calves aged ≤20 days, and the highest infection rate of C. ryanae was seen in calves ≥31 days of age. The occurrence of C. parvum (χ2 = 25.300, P = 0.000) and C. ryanae (χ2 = 18.020, P = 0.001) was significantly associated with the age of the calves. Eleven different subtypes of the IIa family that belonging to C. parvum were recognized via the sequence analyses of the gp60 gene. Except for two (IIaA18G3R1 and IIaA15G2R1) subtypes, nine subtypes were first identified in calves with diarrhea in the KOR. IIaA18G3R1 was the most frequently detected subtype (72.2% of calves), followed by IIaA17G3R1 (5.6%), IIaA15G2R1 (4.2%), IIaA19G4R1 (4.2%), IIaA16G4R1 (2.8%), IIaA17G4R1 (2.8%), IIaA19G3R (2.8%), IIaA14G1R1 (1.4%), IIaA14G3R1 (1.4%), IIaA15G1R1 (1.4%), and IIaA19G1R1 (1.4%) These results suggest that the prevalence of Cryptosporidium spp. is significantly associated with calf age. Furthermore, the findings demonstrate the high genetic diversity of C. parvum and the widespread occurrence of zoonotic C. parvum in pre-weaned calves. Hence, calves are a potential source of zoonotic transmission with considerable public health implications.

Electrolyte Concentrations and Blood Gas Values in Neonatal Calves With Diarrhea

Portable blood analyzers, which recently have been introduced to veterinary medicine, can facilitate immediate identification of sick calves in livestock farms. However, no appropriate standard values exist for neonatal calves; therefore, reference values for adult cattle guide diagnosis and treatment of newborn calves. Our goal was to determine electrolyte, blood chemistry, and blood gas values from healthy calves and compare them to those for diarrheic calves, thus providing useful information for diagnosis and prognosis. We evaluated 193 calves (£1 month old), including those with (n = 88) and without diarrhea (n = 105), using two-tailed, independent t tests after determining normality (Shapiro−Wilk test). Electrolyte measurements in the diarrheic calves included significant decreases in sodium and significant increases in potassium, chloride, and blood urea nitrogen. Strong ion difference (SID), pH, bicarbonate, partial pressure of carbon dioxide, and base excess (BE) were significantly lower in the diarrheic calves (p < 0.001); the anion gap (AG) was significantly higher among diarrheic calves aged 1-10 days (p < 0.001) compared to healthy calves. Our results demonstrate that SID, pH, bicarbonate, and BE correlated strongly with metabolic acidosis, suggesting that these indicators, including AG, can be important tools for evaluating calves’ health status and for providing useful information to diagnose diarrhea.

Molecular detection of pathogenic Escherichia coli strains and their antibiogram associated with risk factors from diarrheic calves in Jimma Ethiopia

Diarrheagenic Escherichia coli are a number of pathogenic E. coli strains that cause diarrheal infection both in animal and human hosts due to their virulence factors. A cross sectional study was conducted between November, 2016 and April, 2017 to isolate and molecularly detect pathogenic E. coli from diarrheic calves to determine the pathogenic strains, antibiogram and associated risk factors in Jimma town. Purposive sampling technique was used to collect 112 fecal samples from diarrheic calves. Conventional culture and biochemical methods were conducted to isolate E. coli isolates. Molecular method was followed to identify virulence factors of pathogenic E. coli strains. Antimicrobial sensitivity patterns of the isolates were tested using the Kirby–Bauer disk diffusion method. A structured questionnaire was also used to collect information from dairy farms and socio-demographic data. The overall isolation rate of E. coli in calves was 51.8% (58/112) (95% CI 42.0–61.0). The occurrence of the bacterium differed significantly by age, colostrum feeding time, amount of milk given per time and navel treatment (P < 0.05). Multivariable analysis revealed that the odds of being infected was significantly highest in calves which fed 1–1.5 L amount of milk per a time (OR 5.38, 95% CI 1.66–17.45, P = 0.005). The overall virulence genes detection rate was 53.5% (95% CI 40.0–67.0). Eleven (19.6%) of eaeA, 6 (10.7%) of Stx1 and 13 (23.2%) of Stx2 genes were detected from calves isolates. Except ciprofloxacillin, all isolates were resistant to at least one drug. Multi drug resistance was recorded in 68.0% (38/56) of calves isolates. Neomycin, 83.3% (25/30), followed by amoxicillin, 53.3% (16/30) were the highest resisted virulence genes. The study demonstrated considerable isolation rate, multiple antimicrobial resistant isolates and high resistant virulent genes in diarrheic calves. It also indicated that the potential importance of calves as source of pathogenic E. coli strains and resistant genes for human diarrhea infection. Improving the hygienic practice of farms and wise use of antimicrobials could help to reduce the occurrence of pathogenic E. coli in farms. Hence, further studies are needed to describe all virulent factors and serotypes associated with the emergence of drug resistant pathogenic E. coli strains in calves.

Comparison of three diagnostic methods in the diagnosis of cryptosporidiosis and gp60 subtyping of Cryptosporidium parvum in diarrheic calves in Central Anatolia Region of Turkey

The aim of this study was to compare three diagnostic methods for the diagnosis of cryptosporidiosis and to detect subtypes ofCryptosporidium parvum by sequences analyses of gp60 gene in diarrheic calves in several herds in Konya province located in Central Anatolia Region of Turkey. Fecal samples were collected from a total of 194 pre-weaned calves (n=158, ≤15 days old, and n=36, 15 to 40 days old), with diarrhoea. For comparative diagnosis, all samples were examined by modified Ziehl-Neelsen staining of fecal smears for the presence of oocyst, nested PCR-RFLP of SSU rRNA and TaqMan qPCR for the detection of Cryptosporidium DNA. A total of 92 (47.4%) and 104 (53.6%) out of the examined samples were found positive by microscopic examination and molecular tools, respectively. The diagnostic sensitivity and specificity of microscopic identification were determined as 88.5% and 100.0%, respectively compared to molecular assays. Cryptosporidium parvum was the only detected species in all positive samples by species-specific qPCR and nested PCR-RFLP assays. Species identifications were further confirmed by sequence analyses of the SSU rRNA PCR products. There was no statistically significant difference in C. parvum prevalence between early pre-weaned calves and calves older than 15 days. The sequence analyses of the gp60 gene of C. parvum isolates revealed a one subtype IIaA13G2R1 belonging to zoonotic family IIa in diarrheic calves

Calf Diarrhea Caused by Prolonged Expansion of Autochthonous Gut Enterobacteriaceae and Their Lytic Bacteriophages

ABSTRACT Neonatal calf diarrhea is a common disease leading to a major economic loss for cattle producers worldwide. Several infectious and noninfectious factors are implicated in calf diarrhea, but disease control remains problematic because of the multifactorial etiology of the disease. Here, we conducted diagnostic multiplex PCR assay and meta-omics analysis (16S rRNA gene-based metataxonomics and untargeted transcriptional profiling) of rectal content of normal and diarrheic beef calves (n = 111). In the diarrheic calf gut, we detected both microbial compositional dysbiosis (i.e., increased abundances of the family Enterobacteriaceae members and their lytic bacteriophages) and functional dysbiosis (i.e., elevated levels of aerobic respiration and virulence potential). The calf diarrheic transcriptome mirrored the gene expression of the bovine host and was enriched in cellular pathways of sulfur metabolism, innate immunity, and gut motility. We then isolated 12 nontoxigenic Enterobacteriaceae strains from the gut of diarrheic calves. Feeding a strain mixture to preweaning mice resulted in a significantly higher level of fecal moisture content, with decreased body weight gain and shortened colon length. The presented findings suggest that gut inflammation followed by a prolonged expansion of nontoxigenic autochthonous Enterobacteriaceae contributes to the onset of diarrhea in preweaning animals. IMPORTANCE Calf diarrhea is the leading cause of death of neonatal calves worldwide. Several infectious and noninfectious factors are implicated in calf diarrhea, but disease control remains problematic because of the multifactorial etiology of the disease. The major finding of the current study centers around the observation of microbial compositional and functional dysbiosis in rectal samples from diarrheic calves. These results highlight the notion that gut inflammation followed by a prolonged expansion of autochthonous Enterobacteriaceae contributes to the onset of calf diarrhea. Moreover, this condition possibly potentiates the risk of invasion of notorious enteric pathogens, including Salmonella spp., and the emergence of inflammation-resistant (or antibiotic-resistant) microbiota via active horizontal gene transfer mediated by lytic bacteriophages.

Susceptibility and virulence profile of Escherichia coli pathotypes isolated from diarrheic and non-diarrheic calves

Neonatal diarrhea is amongst the most frequent diseases affecting calves, leading to damages in milk production. Although Escherichia coli is a commensal microorganism in the gastrointestinal tract, some pathotypes are known to cause high prevalence of diarrhea and food poisoning. The rapidly increasing resistance of bacteria to antimicrobials leads to the research in new, alternative treatment options. The present study aimed at the detection of E. coli pathotypes in newborn diarrheic and non-diarrheic calves, as well as susceptibility tests for antimicrobials and vegetal extracts. Samples were collected from animals located in dairy farms in the state of Minas Gerais, Brazil. The samples were sent for microbiological isolation, genetic identification using PCR and antimicrobial tests. A total of 35 strains from diarrheic animals tested positive for at least one of the virulence genes analyzed: stx1, stx2, eae, bfp and sta. As for the non-diarrheic animals, 9 isolated strains possessed one of or both stx1 and stx2 genes, classifying these non-diarrheic cattle as reservoirs for the STEC pathotype and possible biological vectors of this pathogen. Regarding the susceptibility tests, most isolates displayed resistance to multiple antimicrobial classes. Among the vegetal extracts tested, all isolates tested sensitive to the active ingredient of Salvia officinalis L. (sage). Showcasing an alternative tool to aid in the treatment of pathogenic bacteria.

Virulence Genes, Antibiotic Resistance and Phylotyping of Escherichia coli O157 Recovered from Diarrheic Calves

E. coli O157 is one of the main food-borne pathogens that attributed to the attaching and effacing Shigatoxigenic E. coli pathotype (AE-STEC). The occurrence of E. coli O157 in diarrheic calves investigated through molecular detection of rfbEO157 encoding gene was 8.02% within E. coli strains. Detection of AE-STEC virulence genes in E. coli O157 strains using multiplex PCR showed the presence of eae, stx1, stx2 and ehylA in percentages of 93.3, 73.3, 20 and 13.3%, respectively. The virulence genes profile of E. coli O157 revealed the predominance of eae+stx1 combination in 66.7% of these strains. All E. coli O157 strains exhibited antibiotic multi-resistances with higher resistance (100%) to amoxicillin/clavulanic acid, cefalexin, cefuroxime and tetracycline, while the lowest resistance was detected for gentamicin (40%). Phenotypic resistance to extended spectrum cephalosporins (ESCs) indicated that 60% of these strains were resistant to ceftriaxone and cefotaxime, while 53.3% were resistant to cefquinome. Molecular detection of extended spectrum β-lactamases (ESBLs) encoding genes recorded the superiority of blaTEM gene (100%), whereas the blaSHV and blaCTXM genes were detected in percentages of 40 and 20%, respectively. The genetic profiling of resistance genes revealed the role of blaCTXM gene in ESCs resistance as blaTEM+blaSHV+blaCTXM combination was detected only in ESC resistant strains. Finally, B2 phylogroup was the most prevalent one (80%) within E. coli O157 strains. This implicates diarrheic calves as a source of highly pathogenic multi-resistant E. coli O157 strains.