Molecular identification of Coxiella burnetii in raw milk samples collected from farm animals in districts Kasur and Lahore of Punjab, Pakistan.

Coxiella burnetii is the worldwide zoonotic infectious agent for Q fever in humans and animals. Farm animals are the main reservoirs of C. burnetii infection, which is mainly transmitted via tick bites. In humans, oral, percutaneous, and respiratory routes are the primary sources of infection transmission. The clinical signs vary from flu-like symptoms to endocarditis for humans' acute and chronic Q fever. While it is usually asymptomatic in livestock, abortion, stillbirth, infertility, mastitis, and endometritis are its clinical consequences. Infected farm animals shed C. burnetii in birth products, milk, feces, vaginal mucus, and urine. Milk is an important source of infection among foods of animal origin. This study aimed to determine the prevalence and molecular characterization of C. burnetii in milk samples of dairy animals from two districts in Punjab, Pakistan, as it has not been reported there so far. Using a convenience sampling approach, the current study included 304 individual milk samples from different herds of cattle, buffalo, goats, and sheep present on 39 farms in 11 villages in the districts of Kasur and Lahore. PCR targeting the IS1111 gene sequence was used for its detection. Coxiella burnetii DNA was present in 19 of the 304 (6.3%) samples. The distribution was 7.2% and 5.2% in districts Kasur and Lahore, respectively. The results showed the distribution in ruminants as 3.4% in buffalo, 5.6% in cattle, 6.7% in goats, and 10.6% in sheep. From the univariable analysis, the clinical signs of infection i.e. mastitis and abortion were analyzed for the prevalence of Coxiella burnetii. The obtained sequences were identical to the previously reported sequence of a local strain in district Lahore, Sahiwal and Attock. These findings demonstrated that the prevalence of C. burnetii in raw milk samples deserves more attention from the health care system and veterinary organizations in Kasur and Lahore of Punjab, Pakistan. Future studies should include different districts and human populations, especially professionals working with animals, to estimate the prevalence of C. burnetii.

A prospective cohort study to identify clinical diagnostic and prognostic markers of primary immune thrombocytopenia in dogs.

BACKGROUND: Primary immune thrombocytopenia (pITP) in dogs presents a diagnostic challenge, and clinical markers of severity are lacking. OBJECTIVES: Identify clinicopathologic features that differentiate pITP from secondary ITP (sITP) and markers related to bleeding severity, transfusion, and survival of dogs with pITP. ANIMALS: Ninety-eight thrombocytopenic dogs (58 pITP and 40 sITP). METHODS: Client-owned dogs with platelet counts <50 000/µL were enrolled in a prospective, multi-institution cohort study. History and treatment information, through a maximum of 7 days, was recorded on standard data forms. Bleeding severity was scored daily using a bleeding assessment tool (DOGiBAT). At-admission blood samples were collected for CBC, biochemistry, C-reactive protein concentration, and coagulation panels, and to measure platelet surface-associated immunoglobulin G (PSAIg) and expression of platelet membrane proteins and phospholipids. Dogs with evidence of coincident disease were classified as sITP. RESULTS: No definitive pITP diagnostic test was found. However, pITP cases were characterized by lower platelet counts, D dimer concentrations, and platelet membrane protein expression than sITP cases. Differentiation between pITP and sITP was further enhanced using logistic regression modeling combining patient sex, coagulation profile, platelet count, D dimer, and PSAIg. A second model of pITP severity indicated that low hematocrit and high BUN concentration were associated with non-survival. Low hematocrit at admission, but not platelet count or DOGiBAT score, was associated with transfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Pending validation studies, models constructed from at-admission clinicopathologic findings may improve differentiation of pITP from sITP and identify the most severe pITP cases at the time of presentation.

Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, Pakistan.

Coxiella burnetii is the zoonotic pathogen that causes Q fever; it is widespread globally. Livestock animals are its main reservoir, and infected animals shed C. burnetii in their birth products, feces, vaginal mucus, urine, tissues, and food obtained from them, i.e., milk and meat. There were previously very few reports on the prevalence of C. burnetii in raw meat. This study aimed to determine the prevalence of C.burnetii and its molecular characterization in raw ruminant meat from the Kasur and Lahore districts in Punjab, Pakistan, as this has not been reported so far. In this study, 200 meat samples, 50 from each species of cattle, buffalo, goat, and sheep, were collected from the slaughterhouses in each district, Kasur and Lahore in 2021 and 2022. PCR was used for the detection of the IS1111 element of C. burnetii. The data were recorded and univariate analysis was performed to determine the frequency of C. burnetii DNA in raw meat samples obtained from different ruminant species using the SAS 9.4 statistical package. Of the total of 200 raw meat samples, C. burnetii DNA was present in 40 (20%) of them, tested by PCR using the IS1111 sequence. The prevalence of C.burnetii differed among the studied species of ruminants. When species were compared pairwise, the prevalence in cattle was statistically significantly lower than in sheep (P = 0.005). The sequence alignment based on origin implied that the strains are genetically diverse in different districts of Punjab, Pakistan. The findings demonstrated that the prevalence of C. burnetii, especially in raw meat samples, deserves more attention from the health care system and professionals from Punjab, Pakistan, i.e., abattoir workers and veterinarians.

Effects of pathogen-specific clinical mastitis occurrence in the first 100 days of lactation 1 on future mastitis occurrence in Holstein dairy cows: An observational study.

The objective of this observational study was to estimate effects of clinical mastitis (CM) cases caused by different pathogens (Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., and CM cases with no growth) occurring in the first 100 d in lactation 1, of a dairy cow on the future rate of occurrence of different types of CM during a cow's full lifetime. The outcomes were occurrence of Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., and CM cases with no growth, after the first 100 d of lactation 1, until a cow's removal through death or sale in that or a subsequent lactation. Data, including information on CM cases, milk production, and event dates (including death or sale dates), were collected from 14,440 cows in 5 New York State Holstein herds from January 2004 until February 2014. Generalized linear mixed models with a Poisson distribution and log link function were fit for each pathogen. The individual cow was the unit of analysis. Escherichia coli was a predictor of future occurrence of E. coli, Klebsiella spp., and CM cases with no growth. Early-occurring Klebsiella spp. was a predictor of future cases of Klebsiella spp. Cases with no growth were predictors of future occurrence of Staphylococcus spp., E. coli, Klebsiella spp., and cases with no growth. Thus, E. coli and cases with no growth occurring early in lactation 1 appear to be consistent risk factors for future cases of CM, whether cases with the same pathogen or a different pathogen. In this study, farm effects on later pathogen occurrence differed somewhat, so treatment protocol and culling strategy may play a role in the findings. Nevertheless, the findings may help farmers in managing young cows with CM in early productive life, especially those with E. coli or cases with no growth, in that they may be more susceptible to future CM cases in their later productive life, thus meriting closer attention.

Association of pathogen-specific clinical mastitis in the first 100 days of first lactation with productive lifetime: An observational study comparing competing risks models for death and sale with the Cox model.

The objective of this observational study was to study the association between clinical mastitis (CM) (Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., cases with other treated or other not treated organisms, CM without growth) occurring in a dairy cow's first 100 days (d) of her first lactation and her total productive lifetime, ending in death or sale (for slaughter). Data were collected from 24,831 cows in 5 New York Holstein herds from 2004 to 2014. Two analytical approaches were compared. First, removals (death, sale) were treated as competing events in separate survival analyses, in proportional subdistribution hazards models. In one, death was coded as the event of interest and sale as the competing event; in another, sale was the event of interest and death the competing event. Second, traditional survival analysis (Cox proportional hazards) was conducted. In all models, the time variable was number of days from date of first calving until event (death or sale) date; if the cow was alive at study end, she was censored. Models were stratified by herd. Ten percent of cows died; 48.4 % were sold. In the competing risks analysis, E. coli and CM without growth were associated with death; the former with an increased hazard rate of death, the latter with a lower one. Streptococcus spp., Staph. aureus, Klebsiella spp., cases with other treated or untreated organisms, and CM without growth were associated with higher hazard rates of sale. The Cox proportional hazards model's hazard rates were higher than those in the competing risks model in which death was the event of interest, and resembled those in the model in which sale was the event of interest. Four additional Cox models, omitting dead or sold cows, or censoring each, were also fitted; hazard ratios were similar to the above models. Proportional subdistribution hazards models were appropriate due to competing risks (death, sale); they produce less-biased estimates. A study limitation is that while proportional subdistribution hazards models were appropriate, they have the illogical feature of keeping subjects at risk for the event of interest even after experiencing the competing event. This is, however, necessary in estimating cumulative incidence functions. Another limitation concerns pathogen variability among study farms, implying that CM decisions are farm-specific. Misclassification of 'dead' vs. 'sold' cows was also possible. Nevertheless, the findings may help in optimizing management of cows contracting specific types of CM early in productive lifetime.

Is Salmonella enterica shared between wildlife and cattle in cattle farming areas? An 11-year retrospective study in Tokachi district, Hokkaido, Japan.

BACKGROUND: Salmonella enterica in cattle has long been problematic and suspected to be transmitted by wildlife in Tokachi, Hokkaido, a major cattle farming area in Japan. Understanding the role of wildlife in S. enterica transmission would be helpful for developing control strategies of bovine salmonellosis. OBJECTIVES: We aimed to elucidate the possibility of S. enterica transmission between sympatric wildlife, including raccoons and crows and cattle, in Tokachi from 2008 to 2018 by analysing S. enterica detection records, and the genetic relatedness of serotypes shared between wildlife and cattle. METHODS: S. enterica detection records were based on the results of a field survey and existing cattle records at relevant organisations, including clinical reports, a monitoring survey and quarantine for introduced calves at growing farms and public calving farms. S. enterica was identified by polymerase chain reaction assay and serotyped by agglutination assay. The detection records were organised chronologically to investigate whether common serotypes in wildlife and cattle were detected in the same year. The isolates corresponding to detection records were assessed for their genetic patterns by pulsed-field gel electrophoresis. RESULTS: The prevalence of S. enterica in raccoons and crows was 10.7% (17/159) and 5.7% (55/967), respectively. The following serotypes were detected from both wildlife and cattle: Braenderup, Dublin, Infantis, Mbandaka, Montevideo, 4,[5],12:i:- and Typhimurium. Genetically similar isolates for S. Braenderup, S. Dublin, S. Montevideo and S. 4,[5],12:i:- were detected from both species in the same year. CONCLUSIONS: Our long-term retrospective observations supported that S. enterica was shared between wildlife and cattle. Wildlife invasions should be controlled at farms to prevent inter-species transmission of S. enterica from livestock farms.

The value of pathogen information in treating clinical mastitis.

The objective of this study was to determine the economic value of obtaining timely and more accurate clinical mastitis (CM) test results for optimal treatment of cows. Typically CM is first identified when the farmer observes recognisable outward signs. Further information of whether the pathogen causing CM is Gram-positive, Gram-negative or other (including no growth) can be determined by using on-farm culture methods. The most detailed level of information for mastitis diagnostics is obtainable by sending milk samples for culture to an external laboratory. Knowing the exact pathogen permits the treatment method to be specifically targeted to the causation pathogen, resulting in less discarded milk. The disadvantages are the additional waiting time to receive test results, which delays treating cows, and the cost of the culture test. Net returns per year (NR) for various levels of information were estimated using a dynamic programming model. The Value of Information (VOI) was then calculated as the difference in NR using a specific level of information as compared to more detailed information on the CM causative agent. The highest VOI was observed where the farmer assumed the pathogen causing CM was the one with the highest incidence in the herd and no pathogen specific CM information was obtained. The VOI of pathogen specific information, compared with non-optimal treatment of Staphylococcus aureus where recurrence and spread occurred due to lack of treatment efficacy, was $20.43 when the same incorrect treatment was applied to recurrent cases, and $30.52 when recurrent cases were assumed to be the next highest incidence pathogen and treated accordingly. This indicates that negative consequences associated with choosing the wrong CM treatment can make additional information cost-effective if pathogen identification is assessed at the generic information level and if the pathogen can spread to other cows if not treated appropriately.

Evidence of no protection for a recurrent case of pathogen specific clinical mastitis from a previous case.

The objective of this study was to determine whether the occurrence of a previous case of pathogen-specific clinical mastitis (CM) protects Holstein dairy cows against a recurrent case. Pathogens studied were Escherichia coli, Staphylococcus aureus, Staphylococcus spp., Streptococcus spp., Klebsiella spp., and Trueperella pyogenes. A total of 40 864 lactations (17,265 primiparous and 23,599 multiparous) from 19 835 cows from 5 large, high milk producing New York State dairy herds were analysed. We estimated the effects of parity, calving diseases, milk yield, current season and number of CM cases in the previous lactation on the risk of a first CM case using generalised linear mixed models with a log link and Poisson error distribution. The aforementioned risk factors and the occurrence of previous cases of pathogen-specific CM within the current lactation were evaluated as risks for second and third cases of pathogen-specific CM. Cows with more CM cases in the previous lactation were at greater risk of pathogen-specific CM in the current lactation. Multiparous cows were at greater risk of a second CM case if they had suffered from a first CM case that was caused by the same pathogen as the second case. In contrast, a second CM case generally put cows at greater risk of a third case, irrespective of whether the third case was caused by the same or a different pathogen. Our results showed that a previous case of pathogen specific CM does not generally protect against a recurrent case.

Correlated time to event data: Modeling repeated clinical mastitis data from dairy cattle in New York State.

Mastitis is the most prevalent production disease in dairy herds worldwide and is considered to be the most economically important disease of dairy cattle. Modeling the risk of cows contracting mastitis is therefore of great interest for both targeting prevention programs and evaluating treatment protocols. Clinical mastitis (CM) is a disease of recurrent nature, thus correlation between the subsequent events within one cow may be present. This would violate the assumption behind most statistical time-to-event models. In the case of time to event models, the semi-parametric Cox regression models have become the default tool in modeling the time to an event. Limited methods are currently available to evaluate marginal and random (frailty) effects to account for multiple correlation sources. The objective of this study was to explore the implications of using several Cox or related semi-parametric or parametric models to estimate the hazard for CM in the presence of correlation between events. We evaluated the Andersen-Gill model which uses robust standard errors to account for the correlation, the Conditional Anderson-Gill model that uses stratification to account for event dependence, the Frailty model that introduces a random term to account for unobserved (cow level) heterogeneity, and a related generalized linear mixed model that uses Poisson regression to allow multi-level modeling of time-to-event data. We analyzed data on the occurrence of CM from five dairy farms in New York State. Data were from 8206 cows with 721, 275, 119, and 57 first, second, third, and fourth occurrences of CM, respectively, in the same lactation. The analysis of our sample dataset demonstrated that both cow- and farm-level correlation are present in the case of CM. The Conditional Frailty model was able to model one source of correlation in a random effect and one in a fixed effect. Poisson modeling allowed for simultaneous estimation of within cow correlation and within herd correlation.

How university researchers can contribute to farm-to-table risk assessments: Listeria monocytogenes as an example.

As a part of the Sanitary and Phytosanitary (SPS) agreement, the World Trade Organization requires that member countries establish SPS measures on the basis of an appropriate risk assessment. In addition, many governments use risk assessment in their management of food safety. Consequently, a number of risk assessments for different foodborne pathogens have been conducted. Risk assessments have also been successfully used as a research tool. While, historically, risk assessments are typically initiated by government agencies, university-based researchers are increasingly becoming involved in risk assessments. The purpose of this article is to illustrate the role that university researchers can play in the development and refinement of food safety risk assessments, including possible roles in (1) de novo development or refinement of risk assessment (including farm-to-table risk assessment), (2) data collection supporting risk assessments, and (3) development of new methodological techniques. Transmission of the foodborne pathogen Listeria monocytogenes through the food production chain and risk assessments addressing transmission of this pathogen are used as an example to highlight the roles of university researchers in food safety risk assessments.

Effect of preparing and loading multiple insemination guns on conception rate in two large commercial dairy herds.

In large herds, inseminators frequently thaw multiple straws of semen and prepare several insemination guns at once. The aim of this study was to measure the effect of breeding order, the order that thawed straws are used, on conception rate in commercial dairy herds. A single professional inseminator, utilizing semen from five suppliers, performed 2629 inseminations over 30 months. Up to four straws were thawed at one time with the restriction that straws were used within 20 min of thawing. Straws were thawed per supplier's recommendations, with 66.4% of the straws pocket thawed and the remainder warm-water thawed. Conception was determined by a pregnancy check at 42 days. Data were modeled by multiple logistic regression analysis, which included herd, breeding order, lactation number, times bred, month bred and year bred. Breeding order had no significant effect on the probability of conception: Cows 1-4 achieved adjusted conception rates of 0.36, 0.41, 0.37, and 0.39, respectively. Odds ratios (and 95% confidence intervals) were 1.00 (1.00-1.00), 1.22 (0.99-1.49), 1.04 (0.82-1.32), and 1.12 (0.86-1.45), respectively. Associated laboratory studies, which evaluated the effect of post-thaw holding time on percentage of sperm with progressive motility, found mean values at 20 min holding time to be largely unchanged from mean values at 5 min. Thawing temperatures were 6, and 35 degrees C and holding temperatures were 6, 22, and 35 degrees C. The length of the trial and the wide use of semen from multiple suppliers with differing thawing methods suggests that under similar conditions, a careful and experienced inseminator can thaw multiple straws of semen and prepare insemination guns sufficient to breed up to four cows within 20 min, without an adverse effect on conception.