INTRODUCTION

Paratuberculosis (PTB) or Johne's disease (JD), is a chronic infectious disease that causes granulomatous enteritis in ruminants and eventually other species, with a wide worldwide distribution; caused by Mycobacterium avium ssp. paratuberculosis (MAP) (^{Coelho et al., 2010}). Classified by the World Organization for Animal Health (OIE) as a communicable disease, with socio-economic and public health impact, suggesting the participation of MAP in Crohn's disease in humans (^{OIE, 2017}; ^{Balseiro et al., 2019};^{Gioffré et al., 2015}). Three different strains of MAP have been described: type I (Sheep), type II (Cattle) and type III (Bison or intermediate), according to their preference for infecting different species of ruminants and their different requirements for their bacterial isolation (^{Begg and Whittington, 2010}; ^{Windsor, 2015}). The disease is characterized by signs of progressive emaciation, submandibular edema, and eventually the death of the animals. The main route of infection is fecal-oral, with an incubation period that ranges from one to four years, which is why signs of the disease can be observed from one year of life (^{Chávez et al., 2004}), once MAP enters the intestine, it lodges in macrophages, constituting a granulomatous infection where variable amounts of bacteria can be observed (^{Begg and Whittington, 2010}; ^{Windsor, 2015}). The diagnosis, prevalence and impact of the disease are scarce and inconclusive both in Latin America and in Mexico (^{Chávez et al., 2004}; ^{Begg and Whittington, 2010}; ^{Fernández et al., 2014}).

In Mexico, the presence of PTB in sheep has been detected in various states of the country, such as San Luis Potosí, Guanajuato, Querétaro, Mexico City, the State of Mexico and Veracruz (^{Estévez et al., 2006}). Particularly, in Aguascalientes State, the activity of sheep farming, as in other states of the country, has grown since 1990 by more than 100%, with a meat production of approximately 612 tons in 2018, being the Valle area, made up of the municipalities of Aguascalientes, San Francisco de los Romo, Rincón de Romos, Jesús María, Pabellón de Arteaga and Asientos, where 85% of the State's sheep meat production and livestock activity in general are concentrated (^{SIAP, 2018}).

It is very important to highlight that in this region there are no records of the diagnosis and identification of the diseases that are capable of affecting sheep herds and the adverse effect that these can cause on productivity. In Aguascalientes State there are unquantified cases of adult animals with progressive emaciation and mortality, however, a diagnosis is not made and it is impossible to determine if there is PTB.

Therefore, the objective of this work is to identify the presence of MAP through pathological studies, culture and PCR in sheep with a clinical picture suggestive of PTB, as well as to determine the seroprevalence against MAP by means of ELISA in sheep herds from the Aguascalientes Valley.

MATERIAL AND METHODS

Study design

This descriptive-cross-sectional study was carried out in Aguascalientes State, Mexico, with the herds and study animals selected according to the non-probabilistic method "for convenience" (^{Trusfield, 2018}). In this way, 16 sheep herds were selected with populations ranging between 60 and 340 individuals, making up a total study population of 2,415 animals (Figure 1 and Table 1), where the herd was required to have clinical signs of chronic progressive emaciation suggestive to PTB in at least one specimen older than one year; considering each herd as an experimental unit.

Figure 1 Sheep with signs of emaciation and submandibular edema suggestive of PTB 

Table 1 Paratuberculosis seroprevalence results (ELISA), autopsies performed and bacterial isolation results, by herd in the Aguascalientes Valley 

Herd number	Seroprevalence %	Positive results ELISA-PPA-3	Animals older tan 1 year	Specimens with clinical sings suggestive of PTB /Necropsy performed	Baterial isolation and positive PCR	Location by municipality
14	91.3	168	184	+/1	-	Rincón de Romos
6	84.4	287	340	+/1	+	Aguascalientes (sur)
3	76.3	122	160	+/0	Nr	Rincón de Romos
11	73.1	117	160	+/1	+	Asientos
10	64.4	125	194	+/1	-	Jesús María
9	61.4	72	140	+/1	+	Asientos
4	58.6	99	169	+/1	+	Aguascalientes (sur)
15	56.8	50	88	+/1	-	San Francisco de los Romo C
13	54.5	36	66	+/0	Nr	Pabellón de Arteaga
16	46.7	56	120	+/0	Nr	Aguascalientes (Nte)
1	43.8	35	80	+/1	-	Jesús María
8	28.3	17	60	+/0	Nr	Pabellón de Arteaga
5	12.9	8	62	+/0	Nr	Aguascalientes (Nte)
7	9.5	31	326	+/0	Nr	Aguascalientes (sur)
12	8.1	8	99	+/0	Nr	San Francisco de los Romo
2	4.8	8	167	+/1	+	San Francisco de Los Romo
16	51.3	1239	2415	16/9

Nr: Not perfomed

RESULTS

Seroprevalence. The mean seroprevalence percentage of the 16 herds was 51.3%, with a minimum value of 4.8% and a maximum value of 91.33% with a 95.0% confidence interval for the mean of: [32.7173, 62.3415]. The individual seroprevalence results for each herd are described in Table 1. In the sixteen herds in which the ELISA tests (PPA- 3) were performed, positive cases were identified, obtaining a seroprevalence calculated at “herd level” 100% (Table 1).

Anatomopathological study. In the 9 animals that were necropsied, they presented emaciation, hairy wool or hair, submandibular edema (Figure 1), presence of ascites, fat degeneration, as well as apparent thickening of the intestinal mucosa at the ileum and jejunum (Figure 2), in addition to an increase in the size of the mesenteric lymph nodes and their fusion, not finding evidence of alterations or lesions suggestive of another differential etiology to cases of PTB. Through the histopathological study, it was possible to observe in 8 of the 9 animals studied (88.8%), the presence of enteritis, granulomatous lymphadenitis with the presence of macrophages, giant cells and the presence of countless Acid-Alcohol Resistant Bacteria (ABB) by staining from Ziehl-Neelsen (ZN). Determined as multibacillary lesions (Figure 3). While in 1 of the 9 animals studied (11.2%), the presence of multifocal granulomatous lesions with a low number of bacilli (BAAR) inside macrophages was observed, coinciding with a paucibacillary lesion.

Figure 2 Macroscopic lesions corresponding to intestinal thickening characteristic of granulomatous enteritis 

Figure 3 Histopathological lesions of multibacillary ID, Ziehl-Neelsen stain 400X where multiple BAARs are observed in intestinal mucosa 

Bacterial isolation. After 12 weeks of incubation, in 5 cases (55.5%), development of yellow, creamy, convex and circular colonies characteristic of MAP were observed in L- Jm media added with mycobactin. While in 4 of the cases (44.5%), no colony development was observed until week 40. All the media that were not added with mycobactin did not show growth.

PCR IS900. From the five bacterial isolates obtained, amplification products of the IS900 sequence corresponding to 310 base pairs (bp) were obtained, thus confirming the presence of MAP from these isolates obtained (Figure 4).

Figure 4 Photograph of a 2% agarose gel stained with ethidium bromide. Where amplification products with a size of 310 bp are observed, from DNA of bacterial colonies obtained from intestinal tissue, where the molecular weight marker 100 bp, C + positive control, negative control lane 1 (water ), lanes 3 to 7 amplification product obtained from isolates 03, 04, 05, 06 and 09, which correspond to an IS900 insert fragment of MAP (*). 

Thus, in 100% of the sample (16 study herds) MAP seropositivity was obtained; in turn, in 31.25% of the sample (5 herds) the presence of MAP was confirmed by bacterial isolation and identification by PCR IS900 from the animals that presented clinical symptoms at the time of the study (Table 2).

DISCUSSION

The mean seroprevalence obtained in the present study (51.3%) is reported to be high, compared to other studies conducted to measure prevalence both in the country and worldwide, considering that the disease internationally has a high prevalence in different species of ruminants, with 20% and up to 40% (^{Whittington et al., 2019}). In Mexico, various studies indicate serological evidence for MAP such as those reported by ^{Jaimes et al., (2008)} in San Luis Potosí, Mexico; where the seroprevalence results in sheep by the Agar-gel immunodiffusion method was 44%. While in Nayarit state, 28.94% was reported in sheep, through ELISA tests (^{Mejía et al., 2015}). In Latin America, few studies carried out in sheep show a wide variability in the prevalence of MAP, where prevalences from an average 4.3% in sheep are reported (^{Fernández et al., 2014}), to 54.08% of seroprevalence to MAP in sheep in Brazil, (^{Medeiros et al., 2012}).

Given the few studies, and a wide variation in the methodology and diagnostic test used to assess the prevalence of PTB, ^{Espechit et al. (2017)} and ^{Whittington et al., (2019)} point out that this results in a great variability of the results, and consequently, an underestimation of the presence and true impact of paratuberculosis.

Despite the high frequency results obtained, at the time of the study, only clinical cases (9/16) were found in 56.25% of the herds studied, where all of them show multibacillary or paucibacillary histopathological lesions, characteristic of the disease where if it is considered that these occur in individuals older than one year of age, with a prolonged time of infection coupled with the presentation of a characteristic clinical picture, highly suggestive of PTB, they can be considered a definitive diagnosis of MAP individually, hardly ruled out by other tests such as culture and PCR as mentioned by several authors (^{Pérez et al., 1999}; ^{Dennis et al., 2011}; ^{Windsor, 2015}; ^{Whittingtong et al., 2019}).

Considering that the nine animals that were necropsied presented lesions characteristic of PTB, only in 55.5% (5/9) was bacterial isolation achieved despite presenting multibacillary lesions in eight of the nine. The difficulty of isolating MAP, the culture medium used considering the possible variation of the type of MAP present, "S" or "C", which present a difference in nutritional requirements for growth (^{Elguezabal et al., 2011}; ^{Whittington, 2011}; ^{Stevenson, 2015}; ^{Whittington et al., 2019}), or at the time of the bacterial decontamination process, where the number of viable bacteria for cultivation and isolation can be reduced. The isolation and cultivation of the microorganism in sheep is extremely difficult, being in sheep herds that fecal culture detects fewer than clinical cases and requires more than 12 months for incubation, as reported by ^{Windsor (2015)}, as well as ^{Whittington, 2010}.

The subsequent confirmation and identification of the isolates obtained from MAP by means of IS900 PCR, carried out from the five cultures obtained, confirmed the presence of MAP. Some authors currently suggest that the identification of the bacteria by PCR be carried out from the samples obtained from the tissues, speeding up the time of the results, without directly depending on the cultures obtained, since the diagnostic times are prolonged by the characteristics of MAP isolation, indicating that this test has a similar efficacy with cultures (^{Kawaji et al., 2007}; ^{Nilsen et al., 2008}; ^{Whittington et al., 2019}).

According to authors such as ^{Kumthekar, et al., (2013)}) and ^{Konbon (2018)}, determining the degree of infection of the disease in herds allows establishing adequate control programs that significantly impact the cost-benefit; primarily considering test-scrap. Well, given the infeasibility of discarding and considering the limited availability of some producers for the vaccination of herds, the control measures must be framed towards a strengthening of biosecurity.

Considering thus the early separation of the lambs and the elimination of animals with initial clinical signs of the disease, it may be a measure that positively impacts the reduction of the prevalence of the disease (Robee, 2011; ^{Windsor 2015}). Emphasizing that professionals in the animal health sector and leaders of the productive sector have a clear knowledge of the degree of presence and impact of the disease, both clinically, economically and public health; thus achieving control proposals (^{Roche, 2015}; ^{Windsor, 2015}; ^{Wittington et al., 2019})

CONCLUSION

The presence of Mycobacterium avium ssp paratuberculosis is confirmed as an etiological agent of the clinical disease, in addition to observing the high frequency presence of animals seropositive to MAP, in herds of the Central Valley of Aguascalientes state, Mexico which makes it a high-impact pathology in sheep production in the state.