Prevalence of Coxiella burnetii in bulk milk samples from dairy bovine, ovine, caprine, and camel herds in Iran as determined by polymerase chain reaction

Q fever is a widespread zoonosis caused by the obligate intracellular micro-organism Coxiella burnetii. The objective of this study was to determine the prevalence rate of C. burnetii in bulk milk samples from dairy bovine, ovine, caprine, and camel herds in Isfahan province, Iran. In the present study, 567 bulk milk samples from 186 dairy bovine, ovine, caprine, and camel herds were tested for C. burnetii using a nested polymerase chain reaction assay. The animals whose milk samples collected for this study were clinically healthy. In total, 8 of 247 (3.2%) bovine milk samples were positive; the positive samples originated from 6 of 90 (6.7%) dairy herds. Eight of 140 (5.7%) ovine bulk milk samples from 42 sheep breeding farms and 5 of 110 (4.5%) caprine bulk milk samples from 32 goat breeding farms were positive for C. burnetii. One of 70 (1.4%) camel bulk milk samples from 22 camel breeding farms was also positive for C. burnetii. Although no extensive prevalence study was undertaken, the results of this study indicate that clinically healthy dairy animals are important sources of C. burnetii infection in Iran. To the authors' knowledge, this study is the first report of direct identification of C. burnetii using polymerase chain reaction in bulk milk samples from dairy ovine herds in Iran and the first report of direct identification of C. burnetii in bulk milk samples from dairy camel herds. Further intensive prevalence studies on Coxiella infection and on possible risks of dairy products will be needed to elucidate the epidemiology of Q fever in Iran.     

Genomic diversity and prevalence of Rotavirus in cow and buffalo calves in northern India

Faecal samples were collected from seventy-eight diarrhoeic cow and buffalo calves between November 1998 and February 1999 to study the genomic diversity and prevalence of Rotavirus infection by ribonucleic acid polyacrylamide gel electrophoresis (RNA-PAGE) and enzyme-linked immunosorbent assay (ELISA). In the organised dairy farm (where daily production and health records were maintained), the overall prevalence of infection with Rotavirus, recorded by RNA-PAGE and ELISA, was 27.02% (10/37) in both cow and buffalo calves. In unorganised dairy herds (where no production or health records were maintained), RNA-PAGE and ELISA detected infection with Rotavirus in 26.8% (11/41) of cow and 19.5% (8/41) of buffalo calves. Five distinct electropherotypes were found to circulate in cow and buffalo calves. All were short electropherotypes except the single long electropherotype observed in a buffalo calf in an unorganised dairy herd. Some differences in RNA migration pattern were observed when these electropherotypes were compared with the neonatal calf diarrhoea virus strain of Rotavirus. Some electropherotypes were restricted to one farm while others were found in both organised and unorganised dairy herds and in both cow and buffalo calves.     

The prevalence of Escherichia coli O157.H7 in dairy and beef cattle in Washington State.

Escherichia coli O157.H7 was found in 10 of 3570 (0.28%) faecal samples from dairy cattle in 5 of 60 herds (8.3%). Several tentative associations with manure handling and feeding management practices on dairy farms were identified. Faecal/urine slurry samples, bulk milk samples, and milk filters from dairy herds were negative for E. coli O157.H7. E. coli O157.H7 was also isolated from 10 of 1412 (0.71%) faecal samples from pastured beef cattle in 4 of 25 (16%) herds. The prevalence of E. coli O157.H7 excretion in feedlot beef cattle was 2 of 600 (0.33%). The identification of cattle management practices associated with colonization of cattle by E. coli O157.H7 suggests the possibility that human E. coli O157.H7 exposure may be reduced by cattle management procedures.     

Survey of Aflatoxin M1 in Cow, Goat, Buffalo and Camel Milks in Ismailia-Egypt

Milk from buffalo, cow, goat and camel species was collected in Ismailia in Egypt. Aflatoxin (AFM₁) levels were lower than previous surveys, and were influenced by feeding practices. Cows and buffaloes are fed prepared rations and had highest incidence of AFM₁. Camels forage freely on available pasture and had lowest AFM₁ in their milk. Goats are fed a combination of prepared ration as a supplement to pasture grazing. Most milks (80%, 74%, 66% and 52% of the camel, goat, cow and buffalo milks, respectively) were below the European Union maximum of AFM₁ <50 ng/L and all milk samples were <500 ng/L.     

Characterization of Echinococcus granulosus isolates from human, sheep and camel in Iran.

In the present study, Echinoccocus granulosus isolates collected from human, sheep and camel samples in Iran were characterized based on rostellar hook morphology of protoscoleces as well as PCR-RFLP. Morphological study on human and animal isolates showed the presence of two distinct strains of the parasite, one in sheep and the other one in camels. In this regard, rostellar hook of sheep isolates were significantly different from those of camel origin, meanwhile human isolates were found to be similar to those isolated from sheep. Molecular analysis of the ITS1 region of rDNA derived from human, sheep and camel isolates were in agreement with the morphological findings. Based on the PCR-RFLP method, the sheep and human isolates appeared to pertain to the same genotype and the camel isolates were appeared to pertain to a different genotype.     

Prevalence of Coxiella burnetii (Q fever) antibodies in bovine serum and bulk-milk samples

Q fever (Coxiella burnetii) is a zoonotic disease of increasing public health importance. The objective of this study was to estimate the prevalence of, and risk factors associated with, exposure to C. burnetii in cattle in the Republic of Ireland. Bulk-tank milk samples from 290 dairy herds and 1659 sera from 332 dairy and beef herds, randomly sampled, were tested by indirect ELISA to detect antibodies to C. burnetii. In total, 37·9% of bulk-milk sample herds and 1·8% of sera (from 6·9% of herds) were antibody positive. Of risk factors tested using logistic regression analysis, only large herd size (bulk-milk analysis) and dairy breed (serum analysis) significantly increased the odds of being positive for antibodies to C. burnetii. Herds with positive milk or serum samples were randomly distributed throughout the Republic of Ireland and no clustering was observed. The use of an ELISA to test bulk-milk samples collected by randomized stratified sampling is a cost-effective method by which national herd prevalence can be estimated by active surveillance.     

Reduction of Coxiella burnetii prevalence by vaccination of goats and sheep, The Netherlands

Recently, the number of human Q fever cases in the Netherlands increased dramatically. In response to this increase, dairy goats and dairy sheep were vaccinated against Coxiella burnetii. All pregnant dairy goats and dairy sheep in herds positive for Q fever were culled. We identified the effect of vaccination on bacterial shedding by small ruminants. On the day of culling, samples of uterine fluid, vaginal mucus, and milk were obtained from 957 pregnant animals in 13 herds. Prevalence and bacterial load were reduced in vaccinated animals compared with unvaccinated animals. These effects were most pronounced in animals during their first pregnancy. Results indicate that vaccination may reduce bacterial load in the environment and human exposure to C. burnetii.     

Isolation of Brucella organisms from the milk of seronegative cows

During an investigation of bovine brucellosis in Iran, conducted by the Razi Institute over a twelve-month period, samples of serum and milk were collected simultaneously from 6,472 cows in eight infected herds for serological and bacteriological testing. A total of 1,056 cows were serologically positive and 1,632 of 6,472 milk samples were positive to the milk ring test (MRT). Culture of the positive milk samples yielded 397 isolates of Brucella, 119 of which came from the 5,686 seronegative cows. The isolates belonged to Brucella abortus biotypes 2 (one isolate), 3 (356 isolates) and 9 (40 isolates).     

Seroprevalence of Schmallenberg virus antibodies among dairy cattle, the Netherlands, winter 2011-2012

Infections with Schmallenberg virus (SBV) are associated with congenital malformations in ruminants. Because reporting of suspected cases only could underestimate the true rate of infection, we conducted a seroprevalence study in the Netherlands to detect past exposure to SBV among dairy cattle. A total of 1,123 serum samples collected from cattle during November 2011-January 2012 were tested for antibodies against SBV by using a virus neutralization test; seroprevalence was 72.5%. Seroprevalence was significantly higher in the central-eastern part of the Netherlands than in the northern and southern regions (p<0.001). In addition, high (70%-100%) within-herd seroprevalence was observed in 2 SBV-infected dairy herds and 2 SBV-infected sheep herds. No significant differences were found in age-specific prevalence of antibodies against SBV, which is an indication that SBV is newly arrived in the country.     

Detection of Mycobacterium bovis-infected dairy herds using PCR in bulk tank milk samples

Bovine tuberculosis (bTB) is a chronic and zoonotic disease due to Mycobacterium bovis. The tuberculosis eradication campaign carried out in Argentina has considerably improved the health situation of the herds. Here we evaluated a strategy to detect M. bovis-infected herds by Touch-Down IS6110 polymerase chain reaction (PCR) in bulk tank raw milk from dairy farms. We evaluated 177 samples from herds with the official tuberculosis free certificate (TFC) and 80 from herds without the certificate, non-tuberculosis-free certificate (NTFC), from 10 departments of Santa Fe province, Argentina. To avoid the effect of Taq polymerase inhibitors, a dilution of DNA template was performed. Positive PCR results were obtained in 102 (40%) of the samples, whereas negative ones were obtained in 155 (60%) of the samples. Importantly, 44% of NTFC and 38% of TFC samples were positive. All samples were subjected to culture in L?wenstein Jensen and Stonebrink media with no positive isolation. The negative predictive value (NPV) of PCR in the TFC group was 95%, while the positive predictive value (PPV) of PCR in the NTFC group was 51%. Based on these results, this work proposes a method that should be applied regularly to detect M. bovis--infected dairy herds, complementary to the official test of tuberculin, or purifed protein derivative (PPD), to control dairy herds, especially those free of tuberculosis.     

A Survey of Aflatoxin M1 Contamination in Bulk Milk Samples from Dairy Bovine, Ovine, and Caprine Herds in Iran

A total of 150 bovine (60), ovine (42), and caprine (48) bulk milk samples were analyzed using a commercially available competitive ELISA kit. Overall, AFM1 was found in 46.7 % of the analyzed samples by an average concentration of 40.3 ± 22.2 ng/L. The incidence rates of AFM1 contamination in bovine, ovine, and caprine bulk milk samples were 66.7, 31.0, and 35.4 %, respectively. The concentration of AFM1 in 37.5 % of AFM1-positive bovine milk samples and 5.9 % of AFM1-positive caprine milk samples were higher than 50 ng/L.     

Incidence of Listeria spp. in raw milk in Shahrekord, Iran

Listerosis may be transmitted by direct contact with infected animals or by consumption of contaminated vegetables, meat, or milk products. Raw milk samples obtained from the Milk Industry Foundation, five private dairy companies, and individual dairy farms in Shahrekord, Iran, were tested for the presence of Listeria species. A total of 500 raw milk samples were analyzed by two-stage enrichment techniques. Identification of isolated Listeria was done using the Micro-ID Listeria Kit (Remel, Lenexa, KS). The overall incidence of Listeria species in raw milk was 2.2%. L. monocytogenes was found in 1.6% of the raw milk samples, while L. innocua was found in 0.6% of the samples. Additional studies are needed to assess the public health impact of contaminated milk in Shahrekord.     

A study of the foodborne pathogens: Campylobacter, Listeria and Yersinia, in faeces from slaughter-age cattle and sheep in Australia.

In a study of faeces from 475 slaughter-age cattle and sheep from 19 herds or flocks, Campylobacter species (C. jejuni and C. coli) were cultured from all production systems studied and from 73.7 per cent (14/19) of herds or flocks. Within individual properties there was a higher prevalence in cattle than in sheep, with Campylobacter being most commonly isolated from feedlot cattle. The median prevalences and ranges were: for dairy cattle, six per cent (0-24%), feedlot beef cattle, 58 per cent (12-92%) pasture beef cattle, two per cent (0-52%), mutton sheep, 0 per cent (0-4%) and prime lambs eight per cent. Listeria ivanovii was cultured from one dairy cow but Yersinia enterocolitica was not cultured from any animal. Campylobacter is the leading bacterial causative agent of acute diarrhoea in humans in many industrialised countries. While the role of cattle and sheep in producing human campylobacteriosis either directly or via contaminated food, remains to be epidemiologically clarified, this study suggests that the production system, particularly for cattle, may be an important consideration.     

Coxiella burnetii in bulk tank milk samples, United States

Dairy cattle are a primary reservoir of Coxiella burnetii, which causes Q fever. However, no recent nationwide studies have assessed the prevalence and risks of Q fever in dairy cattle. We report >or=94% prevalence in samples of bulk tank milk from U.S. dairy herds tested during the past 3 years.     

Brucellosis in camels, cattle and humans: associations and evaluation of serological tests used for diagnosis of the disease in certain nomadic localities in Sudan

Brucellosis was studied in 2,225 camels, 20 camel nomads and 33 abattoir workers in certain nomadic localities in Sudan, using serum and milk samples. Lymph nodes, testicular tissues and udder tissues from positive camels and hygroma aspirates from three affected cows were used for isolation of Brucella. Serum samples were examined by Rose Bengal plate test (RBPT), modified RBPT (mRBPT), serum agglutination test (SAT) and competitive enzyme-linked immunosorbent assay (cELISA), and milk by the milk ring test. Overall seroprevalence in camels (milk and serum samples) was 37.5%. The seroprevalence in males was 28.2% and in females 40.1%. Twelve (60%) of the 20 nomads and three (9%) of the 33 abattoir workers had positive antibody titres. Brucella abortus biovar 6 was isolated from two camels and three cows. Two isolates, one from each species, were atypical. The bacteriological findings suggested that camels were infected from cattle, the primary hosts of B. abortus. The mRBPT was suitable for screening camel sera for brucellosis, but the cELISA detected 2.1% more positives. The SAT antibody concentrations ranged between < 13 and 3,282 IU/ml.     

Campylobacter jejuni in dairy cows and raw milk

Twelve herds of dairy cows were examined by rectal swabbing for the presence of Campylobacter jejuni. Ten herds were positive with the incidence of colonized animals ranging from 10 to 72% of those tested. With the exception of the two negative herds where mains water only was consumed, all animals drank from rivers or streams when grazing. There was no relationship between total and coliform counts and the presence of C. jejuni in raw milk. However, milk from one farm that consistently gave positive results had significantly higher Escherichia coli counts than other samples.     

Optimization of Serum EVELISA for Milk Testing of Johne's Disease

Abstract Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most economically important diseases of dairy cattle. Control of JD could be achieved by good herd management practices, and diagnosis; however, this approach has been hampered by the low sensitivity of currently available enzyme-linked immunosorbent assay (ELISA) tests. In our previous study, we developed a sensitive serum ELISA test, ethanol-vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti-MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD-free herds. We evaluated five environmental mycobacteria as absorbents of cross-reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal polymerase chain reaction test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean±SD=0.355±0.455) was significantly higher than that in the JD-negative milk samples (mean±SD=0.071±0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.     

Molecular Characterization of Echinococcus granulosus from Hydatid Cysts Isolated from Human and Animals in Golestan Province,North of Iran

Background

The aim of the present study was to determine the molecular characteristics of Echinococcus granulosus from paraffin-embedded tissues of hydatid cysts isolated from human and protoscoleces of hydatid cysts from sheep, cattle and camel isolates using PCR- RFLP of ITS1- rDNA analysis in Golestan Province, northern Iran.

Methods

E. granulosus isolates from human patients infected with hydatid cyst and protoscoleces from hydatid cysts of sheep, cattle and camel isolates were collected from different hospitals and the abattoir throughout the Golestan Province. In all, 60 E. granulosus genomic DNA were extracted and examined by PCR - ITS1 of rDNA and amplified using BD1 / 4S and EGF1 / EGR2 primers, followed by RFLP using Alu1, Msp1 and TaqI restriction enzymes.

Results

The PCR-ITS1 products obtained from sheep, cattle and human isolates were similar to sheep strain (1000 bp and 391 bp). Majority of the camel samples yielded 295 bp DNA bands. RFLP -ITS1 of E. granulosus with Taq1 in human, sheep and cattle isolates showed similar patterns in the number and size of DNA. RFLP methods in camel isolates showed a different genotype, using Taq1, whereas no DNA bands were observed using Alu1 in camel and human isolates. Therefore, two clearly distinguishable banding patterns of E. granulosus were obtained with the three enzymes, which separating human, sheep and cattle isolates from the camel origin.

Conclusion

The results indicate the possible of transmission of the G1 and G6 genotypes of E. granulosus between livestock animals and human in Golestan Province.     

Molecular typing of Giardia duodenalis in cattle,sheep and goats in an arid area of central Iran

Giardia duodenalis is one of the most common intestinal parasites in humans as well as livestock and wildlife. It is of both public and veterinary health importance in developing nations. A molecular survey of Giardia duodenalis assemblages in ruminants from Yazd Province, Iran was conducted on 484 animal faecal samples collected per rectum from slaughtered ruminants including 192 cattle, 192 sheep and 100 goats from June to November 2017. Species-specific and assemblage-specific PCRs for assemblages A, B and E at the triose phosphate isomerase (tpi) gene were performed, and samples positive for Giardia were confirmed by sequencing. In total, 25 (5.16%) of examined faecal samples including eight cattle (4.2%), twelve sheep (6.2%) and five goats (5%) were infected with G. duodenalis. Assemblage-specific PCR detected G. duodenalis assemblage E in seven faecal samples (six in sheep and one in a goat). Assemblages A and B were not detected. This study provides the first insight into Giardia infection in slaughtered livestock in Iran. Although the prevalence of infection with Giardia in this hot-arid area of Iran was low, educating people about direct contact with livestock such as farmers and abattoirs workers about this zoonotic infection is important.     

Detection of sorbitol-negative and sorbitol-positive Shiga toxin-producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, and Salmonella spp. in dairy farm environmental samples

Six visits were conducted to four dairy farms to collect swab, liquid, and solid dairy farm environmental samples (165 to 180/farm; 15 sample types). The objective of the study was to determine on-farm sources of Campylobacter jejuni, Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC), which might serve as reservoirs for transmission of pathogens. Samples were analyzed using mostly U.S. Food and Drug Administration's Bacteriological Analytical Manual protocols; however, Salmonella spp., L. monocytogenes and STEC were co-enriched in universal pre-enrichment broth. Campylobacter jejuni were enriched in Bolton broth containing Bolton broth supplement. Pathogens were isolated on agar media, typed biochemically, and confirmed using multiplex polymerase chain reaction protocols. Campylobacter jejuni, Salmonella spp., L. monocytogenes, Sorbitol-negative (SN)-STEC O157:H7, and sorbitol-positive (SP)-STEC, respectively, were isolated from 5.06%, 3.76%, 6.51%, 0.72%, and 17.3% of samples evaluated. Whereas other pathogens were isolated from all four farms, SN-STEC O157:H7 were isolated from only two farms. Diverse serotypes of SP-STEC including O157:H7, O26:H11, O111, and O103 were isolated. None of the five pathogen groups studied were isolated from bulk tank milk (BTM). Most pathogens (44.2%) were isolated directly from fecal samples. Bovine fecal samples, lagoon water, bedding, bird droppings, and rat intestinal contents constituted areas of major concern on dairy farms. Although in-line milk filters from two farms tested positive for Salmonella or L. monocytogenes, none of the pathogens were detected in the corresponding BTM samples. Good manure management practices, including control of feral animals, are critical in assuring dairy farm hygiene. Identification of on-farm pathogen reservoirs could aid with implementation of farm-specific pathogen reduction programs.