EFFECTS OF SUBCLINICAL MYCOBACTERIUM AVIUM SSP . PARATUBERCULOSIS INFECTION ON SOME PHYSIOLOGICAL PARAMETERS , HEALTH STATUS AND PRODUCTION IN DAIRY COWS

Department of Animal Hygiene, Herd Health and Veterinary Ethology, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary; Department of Nutrition, Faculty of Agricultural and Environmental Sciences, Szent István University, Gödöllő, Hungary; Institute of Animal Husbandry, Faculty of Agricultural and Environmental Science, Szent István University, Gödöllő, Hungary; M.A.H Food-Control Ltd., Vet-Control Laboratory, Budapest, Hungary

Paratuberculosis (Johne's disease, JD) is the chronic inflammation of the colonic mucosa in ruminants, caused by Mycobacterium avium ssp.paratuberculosis (MAP).The pathogen can remain viable and infective for up to 12 months in a dry, fully shaded environment (Donat et al., 2016).Wild animals can also be Acta Veterinaria Hungarica 64, 2016 infected and serve as reservoirs (Garcia et al., 2008).According to a recent review (Garcia and Shalloo, 2015), the apparent prevalence in dairy herds is reported be 0.52-41.4% on animal level and 7-83.3% on herd level.True prevalence estimates are rarely obtained.
The disease usually develops to the clinical stage in the second or third lactation, with extremes of 4 months to 15 years of age (Garcia and Shalloo, 2015).The agent can be transmitted with faeces and milk, even in the subclinical phase of the disease (Beaudeau et al., 2007).Airborne infection via dust particles was also described (Eisenberg et al., 2011).Barn hygiene, especially in the calving pen, is crucial in the prevention of new infections (Sweeney et al., 2012;Donat et al., 2016).
Research suggests that MAP might present a zoonotic risk due to its potential association with Crohn's disease in humans (Chiodini et al., 2012).Implementing control programs to reduce spread within and between herds are therefore of fundamental importance (Geraghty et al., 2014).The most common methods in JD monitoring are faecal PCR assays, which proved to be more sensitive than ELISA (Nielsen et al., 2002).
The disease causes serious economic loss to the dairy industry, such as decreased milk yield, hindered feed conversion, lower fertility, and other disorders (Garcia and Shalloo, 2015).Reduced slaughter weight and early culling are also reported (Fodor et al., 2014;Pieper et al., 2015).Several studies have estimated the farm-level economic loss of MAP infections to be in the range of EUR 35-165 per cow (Stott et al., 2005;Tiwari et al., 2008;Fodor et al., 2014).
The aim of present study was to examine the effects of subclinical MAP infections on the health as well as on some clinical biochemical parameters and production traits of high-yielding dairy cows in a Hungarian dairy herd.

Screening of dairy herds
Bulk-tank milk samples were collected for PCR assay in 2014 and 2015 from 29 large-scale dairy farms in Hungary to screen the MAP status of the herds.The only farm with a positive sample was chosen for further studies.
Pooled faecal samplings were performed with five animals/pool, and faecal samples of the animals in the positive pools were then analysed individually.
For DNA extraction, the NucleoSpin ® Tissue Kit (Macherey-Nagel, Germany) was used.The fractions were analysed through real-time PCR (RT-PCR) using the MX3000P of Stratagene RT-PCR system (Thermo Fisher Scientific, USA), and for the detection of MAP Adiavet™ ParaTB Real Time PCR test (bioMérieux, France) was used.
Acta Veterinaria Hungarica 64, 2016 Experimental animals, sampling protocol and data collection Health and production parameters were monitored around the time of calving and through the first semester of lactation and cows in the dry period were also involved in the experiment.A total of 15 cows that were MAP positive by PCR were allocated in the experimental group but none of them showed clinical signs of paratuberculosis, and 15 cows that were negative for both faecal PCR and clinical signs were selected as controls.At the end of the experimental period the MAP status of the cows was re-evaluated.Five animals from the control group were found to be MAP positive, and reallocated in the experimental group.The statistical analysis therefore involved 20 animals as MAP shedders (MAP+, age: 5.6 ± 1.6 years; lactation: 3.5 ± 1.4; BCS: 3.0 ± 0) and 10 animals as control (CO, age: 5.2 ± 2.8 years; lactation: 3.4 ± 2.2, BCS: 3.5 ± 0.3).
Experimental and control cows were not housed or treated separately from other animals during the period of investigation.The housing and feeding conditions were identical for MAP+ and control cows.During lactation the animals were fed total mixed ration (TMR) twice a day and milked four times daily.Drinking water was available ad libitum.
Blood and urine samples were taken between January and July 2015, from day 10 prior to the expected date of calving until day 120 of lactation (Table 1).Blood and urine samples were taken 3-5 h after the morning feeding.Plasma samples were analysed for total protein (TP), albumin, total cholesterol (CH), triglyceride (TG), beta-hydroxybutyrate (BHB), nonesterified fatty acids (NEFA), urea, total calcium, inorganic phosphate, and carotene concentrations.The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) were also measured.These analyses were performed with a BMG Labtech SPECTROstar Nano (BMG Labtech, Germany) biochemistry analyser using commercial kits (Diagon Ltd., Hungary for BHB, Randox Ltd., Ireland for NEFA, and Diagnosticum Ltd., Hungary for all others).
The pH of the urine samples was measured with a digital pH meter (Radelkis OP-211/1, Radelkis Co., Hungary), and net acid-base excretion (NABE) was determined according to the method of Kutas (1965).
At the time of the blood samplings the body condition of the animals was scored (BCS) on a 1 to 5 scale (Mulvany, 1977).Rumen fill and faeces consistency (Zaaijer and Noordhuizen, 2003) were also scored at the same time.
Monthly test-day milk recording data and certain health and reproductionrelated parameters were collected throughout the lactation and obtained from the farm database (Riska™ by Systo, Hungary).

Statistical analysis
Statistical analysis was performed using the R 3.2.3statistical software (R Core Team, 2015).For the analysis of production traits, the Shapiro-Wilk test was used to test the normality of data, and in case of normal distribution a paired t-test, while in case of non-normality the Wilcoxon rank sum test was used to test the difference between groups.For the metabolic parameters, BCS, rumen fill and faeces scores a general linear model was used to calculate the effects between groups, individuals or samplings.Pearson correlations between some parameters were also established.The level of significance was set to P < 0.05.

Metabolic parameters
The results of the blood and urine parameters, body condition, rumen fill and faeces scoring are shown in Table 2.
The body condition, rumen fill and faeces scores differed significantly between the two groups, all of them being lower in the MAP+ cows.There was no significant difference in the plasma or urine metabolic parameters between the groups.There were moderate but significant correlations between BCS and rumen fill (r = 0.23, P = 0.001) as well as between BCS and faeces score (r = 0.41, P < 0.001).The correlations between BCS and BHB (r = -0.14,P = 0.059) or NEFA (r = 0.07, P = 0.307) were low and not significant.

Production and health-related parameters
The results obtained on production and health-related parameters are summarised in Table 3. Milk yield and lactose content tended to be lower (P = 0.074 and 0.077, respectively), somatic cell count (SCC) tended to be higher (P = 0.097), while milk fat content was significantly higher (P = 0.006) in MAP+ cows than in the controls.However, milk protein content did not differ between the groups.
All other health and production parameters, except for pregnancy rate to the first artificial insemination (AI), were significantly better in the CO group.Rónai et al. (2015) reported a 2.4-14.1% animal-level prevalence of MAP infections in Hungary.MAP is present throughout the country in cow populations, and also in wild animals (wild boar, red deer, red fox, water buffalo).The bulk tank milk positivity, as detected by PCR, varies among studies (Sweeney et al., 2012).Khol et al. (2013) found no MAP-positive bulk tank milk samples in their study, while others reported a rate similar to our results (3.4%; Beaver et al., 2016), or even as high as 52% positive samples (Stabel et al., 2002).

Discussion
Subclinical infections usually remain undiagnosed.Animals without clinical signs are not culled and continue to spread the pathogen, thus increasing the infection rate within the herd (Clarke, 1997).Additionally, many MAP-infected animals can show a latent or intermittent shedding stage where no MAP isolated can be found using the current diagnostic methods (Schukken et al., 2015), as it was seen in our study, namely that some MAP-negative animals were rearranged to the MAP+ group at the end of the experimental period.
In the present study there was no difference in metabolic parameters between MAP+ and control cows.This finding was rather surprising, since an impaired metabolic status was presumed in the MAP+ group, especially as there were differences between the two groups in BCS, rumen fill and faeces scores (Table 2).At least some level of hypoproteinaemia or signs of energy imbalance were hypothesised.We suppose that this finding is due to the early stage of MAP infection without any morphologic changes in the gut mucosa.Limited information is available about the metabolic status and changes in blood or urine biochemical parameters in cows shedding MAP.Contrary to our results, Donat et al. (2014a) reported a decreased total protein (TP) level in the serum samples of MAP+ cows, including those shedding a low number of bacteria and presumably being in a subclinical phase of paratuberculosis.In conformity with our results, they did not find differences in BHB, bilirubin and cholesterol levels and ALT activity in MAP-shedding cows.Earlier studies also reported lower levels of TP in subclinically infected calves (Szilágyi et al., 1989;Körmendy et al., 1990).McGregor et al. (2015) reported that several biochemical parameters, except serum albumin, were similar between MAP-positive and MAP-negative Merino sheep.The serum albumin concentration was lower in sheep having more serious histopathology scores and lower body weight.The clinical cases were not excluded in their study, and serum albumin level was similar in sheep with mild (1 and 2) intestinal lesions (maybe subclinical cases) as in the controls.Fodor et al. (2014) reported a higher rate of early culling among MAP+ cows, mostly due to metabolic problems; however, this finding was not verified by the results of blood samplings and biochemical analysis.
The acceptable BCS of cows at the time of calving is 3.0-3.5 (on a 5-point scoring scale), which is expected to decrease by one point in the early milk production period (Roche et al., 2009).Body condition scores started from the ideal 3.0-3.5 at calving in our study, but the rate of decrease of body condition was different in the two groups (Fig. 1), possibly indicating the effect of MAP infection.It has also been reported that JD causes a significant body weight loss in cows (Fodor et al., 2014) andsheep (McGregor et al., 2015); yet, to the best of our knowledge, there are no data about the effect of subclinical MAP infection on the BCS in dairy cows.The faeces score was lower in MAP-shedding animals, although diarrhoea was not observed in any of the animals examined.We found that the lowest faeces scores were obtained at the time of the third sampling (D 10 postpartum, data not shown), but this was possibly caused by the relatively high ratio of concentrate in the TMR at the onset of lactation.Based on these findings it can be suggested that feed conversion -and primarily that of the readily fermentable carbohydrates -is impaired in MAP+ animals, resulting in more liquid faecal consistency.However, further studies are needed to confirm this assumption.
All of the examined reproduction parameters differed between the groups, which suggests that even a subclinical MAP infection can significantly hinder the reproductive success of the herd.In the CO group, the reproduction parameters were better than the herd average, while the MAP+ group revealed worse values.Similarly, in the study of Johnson-Ifearulundu et al. (2000) the calving interval was on average 27.9 days longer in MAP+ cows than in the control animals.
The number of reproductive tract treatments was higher in the MAP+ cows in our study, resulting in higher veterinary and treatment costs in the case of JD (Benedictus et al., 1987;Fodor et al., 2014), although we did not quantify the economic effects of subclinical MAP infection.
According to the study of Weiss et al. (2006), there is a general hyporesponsiveness of the cellular immune system in MAP-infected cows.Antibody production is also severely decreased in the infected animals (Kreeger et al., 1991).Due to the impaired cellular immune response, subclinical paratuberculosis makes animals more susceptible to infections.MAP infection and immunodeficiency may cause secondary diseases, including reproductive and udder problems, that often lead to the culling of animals (Johnson-Ifearulundu and Kaneene, 1997).
The milk yield tended to be lower in the MAP+ group.There are several studies analysing the effects of MAP infection on the milk yield of dairy cows where a decrease in milk yield is reported (Benedictus et al., 1987;Wilson et al., 1993;Nordlund et al., 1996).The decrease in the milk production of subclinically infected cows tends to be progressive, but statistical differences may only be confirmed in the fourth or later lactations (Tiwari et al., 2007(Tiwari et al., , 2008)).However, other authors did not find any difference (Johnson et al., 2001) or even reported an increased milk yield (McNab et al., 1991) in MAP-infected cows.According to a recent meta-analysis by McAloon et al. (2016), the calculated combined effect of MAP infection was -1.87 kg milk/cow per day, estimated to correspond to a 5.9% decrease in milk yield, which is lower than the results of the present study.According to Donat et al. (2014b), the decrease in milk production of MAP-positive cattle depends on the within-herd prevalence.
The higher milk fat percent in the MAP+ group cannot be explained on the basis of the known pathogenesis of the disease and the hypothesised digestive and metabolic disorders.The effect of infection on milk composition is not fully understood.Gonda et al. (2007) found that MAP+ cows produce less milk fat and protein, while Pillars et al. (2011) and Donat et al. (2014b) could not demonstrate such an effect.
The udder health of MAP+ cows is reflected in the number of udder treatments and the higher tendency of somatic cell count in this group.The number of treatments was 4.8-fold higher in the MAP+ cows in the first 120 days of lactation.Several studies have demonstrated that chronic mastitis is one of the main causes of early culling of MAP+ dairy cows (Dufour et al., 2004;McSpadden et al., 2013;Garcia and Shalloo, 2015); however, other authors found no correlation between MAP status and SCC (Gonda et al., 2007;Donat et al., 2014b).

Acta Veterinaria Hungarica 64, 2016
These contradicting results possibly derive from the inaccuracy or alternative application of diagnostic methods (Pillars et al., 2011).If higher numbers of animals are shedding the pathogen, more pronounced effects can be found in the production traits (Lombard et al., 2005;Raizman et al., 2009).Production loss is more severe in animals positive by faecal PCR than in those positive by ELISA (Gonda et al., 2007).According to Smith et al. (2016), low-pathology animals (having only at least one positive culture or positive ELISA result) were shown to recover some productivity, while high-pathology animals (at least one highpositive culture) continued to exhibit a production decrease.
It can be concluded that MAP infection, even in a subclinical form, has a significant impact on the production, some reproductive parameters and the udder health of cows.A more exact estimation of its economic impact might provide an insight into the cost efficiency of screening for subclinical infections, which would help prevent new infections and improve herd health and productivity.

Fig. 1 .
Fig. 1.Body condition score of the cows examined

Table 1
Sampling schedule followed during the experiment CO = control; pp.= postpartum

Table 2
Mean (± SD) values of metabolic parameters, body condition score (BCS), rumen fill and faeces scores in MAP-shedding (MAP+) and control (CO) groups