Veterinary Parasitology 216 (2016) 101–107

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Short communication

Naturally acquired bovine besnoitiosis: Differential distribution of parasites in the skin of chronically infected cattle G. Schares a,∗ , M.C. Langenmayer b,c , M. Majzoub-Altweck b , J.C. Scharr d , A. Gentile e , A. Maksimov a , S. Schares a , F.J. Conraths a , N.S. Gollnick d a

Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Greifswald – Insel Riems, Germany Institute of Veterinary Pathology at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität München, Munich, Germany c Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-Universität München, Munich, Germany d Clinic for Ruminants with Ambulatory and Herd Health Services at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität München, Oberschleißheim, Germany e Department of Veterinary Medical Sciences, University of Bologna, Via Tolara di Sopra 50, Ozzano Emilia, Bologna, Italy b

a r t i c l e

i n f o

Article history: Received 21 October 2015 Received in revised form 17 December 2015 Accepted 18 December 2015 Keywords: Besnoitia besnoiti Skin Tachyzoite Bradyzoite Real-time PCR Diagnosis

a b s t r a c t Bovine besnoitiosis is caused by Besnoitia besnoiti, an apicomplexan parasite closely related to Toxoplasma gondii and Neospora caninum. In the acute stage of besnoitiosis, cattle suffer from pyrexia, swollen lymph nodes, anorexia and subcutaneous edema. In the chronic stage, tissue cysts are formed in a variety of tissues including the skin. Knowledge about the distribution of tissue cysts of different parts of the skin of infected animals is scarce. Four chronically infected cattle were euthanized and skin samples were taken from a total of 77 standardized cutaneous locations per animal. Portions of the dermis were taken, from which DNA was extracted and examined by real-time PCR. Cycle of transition (Ct) values reflecting the amount of parasite DNA in the samples were determined. For statistical analysis, samples were attributed to 11 larger skin regions (‘OuterHindlegDistal’, ‘Rump, ForelegMiddle’, ‘NoseFrontEars’, ‘CheekEye’, ‘SideLowerPart’, ‘ForelegDistal’, ‘SideUpperPart’, ‘LegsInner’, ‘VentralHeadNeck’, ‘DorsalNeckWithersBackTail’). While all samples revealed a positive result in three female cattle, only 63.6% (49/77) of the samples of a bull showed positive results. For statistical analysis, a Ct value of 45 was assumed for samples with a negative result. The dams showed median Ct values of 16.1, 17.5 and 19.4, while in skin samples of the bull a median Ct value of 37.6 was observed. To determine the differences in DNA concentrations between different locations of the skin of the animals, a relative Ct (relCt) was determined by subtracting for each animal indv the MedianCtindv from each sample Ct. Analyses of the relCt values showed that the highest relative parasite DNA concentrations were observed in the categories ‘OuterHindlegDistal’, ‘Rump’, ‘ForelegMiddle’ and ‘NoseFrontEars’. The relCt values in these categories differed statistically significantly from those determined for the categories ‘VentralHeadNeck’ and ‘DorsalNeckWithersBackTail’. The analysis showed clear differences in the distribution and the detectability of parasite DNA in the skin of cattle infected with B. besnoiti. In all four animals, samples from the ‘Rump’ region (Regio fermoris) showed high parasite DNA concentrations. Because this region is also easily accessible for veterinarians, this skin location appears to be optimal for taking skin biopsies for detection or isolation of B. besnoiti. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Besnoitia besnoiti—the cause of bovine besnoitiosis—is a cyst forming apicomplexan parasite closely related to Toxoplasma gondii and Neospora caninum (Ellis et al., 2000). In acutely infected cattle, bovine besnoitiosis is characterized by pyrexia, swollen lymphn-

∗ Corresponding author. Fax: +49 38351 71174. E-mail address: gereon.schares@fli.bund.de (G. Schares). http://dx.doi.org/10.1016/j.vetpar.2015.12.016 0304-4017/© 2015 Elsevier B.V. All rights reserved.

odes, nasal and ocular discharge, salivation, stiff gait and—in severe cases—clinically apparent subcutaneous edema. In chronically infected cattle, the skin may become severely lichenified and alopecic. Bulls may become infertile due to orchitis (Bigalke and Prozesky, 2004; Kumi-Diaka et al., 1981). However, only a few cattle in affected herds develop typical clinical signs, while most animals remain subclinically infected (Alvarez-Garcia et al., 2013; Bigalke, 1968; Jacquiet et al., 2010). In the acute stage of the infection, tachyzoites multiply mainly in endothelial cells of blood vessels (McCully et al., 1966). Later on, characteristic tissue cysts establish, which

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are surrounded by hyaline material consisting of multiple layers of host cell-derived collagen fibrils, thus forming a secondary cyst wall (Dubey et al., 2013; Majzoub et al., 2010). Tissue cysts are located in the skin, the scleral conjunctivae, the mucosal membrane of the vestibulum vaginae and the upper respiratory tract (Basson et al., 1970; McCully et al., 1966). In addition, tissue cysts are regularly found in the aponeuroses of muscles and in fasciae (Basson et al., 1970; Gentile et al., 2012; McCully et al., 1966). Interestingly, internal organs are only rarely affected (Frey et al., 2013). The reasons for these differences in the localisation of tissue cysts of B. besnoiti are poorly understood. Knowledge on the life cycle of B. besnoiti is incomplete, but it was shown that the parasite can be transmitted mechanically by biting flies (Bigalke, 1968). Although it is suspected that a carnivorous definitive host exists it has not been identified so far (Basso et al., 2011; Diesing et al., 1988). Introduction of infected cattle into naive herds seems to play a major role in the transmission of the infection among herds (Bigalke, 1968).

PCR techniques have been developed (Cortes et al., 2007, 2006b; Schares et al., 2011), which have been shown to be more sensitive in the detection of acute and chronic cases than other techniques, including serological and histological methods (Cortes et al., 2006a, 2007; Schares et al., 2013). Although serological tests are easy to perform and less expensive when compared to PCR, they may cause false positive results due to limited specificity (Garcia-Lunar et al., 2015; Nasir et al., 2012). This makes confirmatory tests necessary, such as direct parasite detection. In addition, direct detection methods are important in the diagnosis of chronically infected cattle, because some persistently infected animals do not develop antibodies and can therefore not be detected by serological techniques (Garcia-Lunar et al., 2013; Schares et al., 2010). A recent field study reported tissue cysts in a large number of serologically negative animals (Gutierrez-Exposito et al., 2015). Another study revealed negative PCR results in samples collected from head or ventral skin regions, although the animals had tested serologically positive (Basso et al., 2013). To investigate these diagnostic problems

Fig. 1. Locations of skin samples and graphical summary of the real-time PCR results on skin samples of four cattle with chronic besnoitiosis. Most samples taken from regions including dorsal neck, withers, back and tail were taken from both sides of the median line and are either indicated by ‘R’ (right side) or ‘L’ (left side). For the remaining sites, samples from the right or left side of the animal are documented by two different sample numbers separated by a slash. From each sample Ctindv value, the animal-specific skin median Ct value (MedianCtindv , Table 3) was subtracted and relCt values calculated (Supplementary Table S2). The color code represents regions with Median-relCtreg values ≤−1.0 (red; regions A, B, C), >−1.0 and ≤−0.5 (orange; region D), >−0.5 and ≤0.0 (yellow; regions E, F, G), >0.0 and ≤0.5 (green; regions H, I), >0.5 (blue; regions J, K) (Table 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Animals with chronic bovine besnoitiosis sampled in 77 standardized skin locations. Animal (Herda of origin; reference)

Necropsy date (age in months)

Date of acute Last IFAT titer signs of noted (date) besnoitiosis/seroconversion

Clinical signs

Bull996 (BbGer1; unpublished)

14.04.2011 (102)

18.8.2010

1:3200 (14.4.2011)

SA20 (BbGer1; Schares et al., 2013; Langenmayer et al., 2015a

07.06.2010 (63)

21.8.2009

1:3200 (18.3.2010)

SA22 (BbGer1; Schares et al., 2013; Langenmayer et al., 2015a

13.04.2010 (57)

7.10.2009

1:6400 (30.3.2010)

CowItaly (BbItaE; Gentile et al., 2012

01.07.2010 (36)

11.2009

1:12800 (25.5.2010)

Prior to slaughter the bull presented tissue cysts in the scleral conjunctivae and tissue cysts in prepuce mucosa; Skin: focal alopecia at regio colli lateralis, regio scapularis, regio thoracis, regio abdomnalis lateralis and regio femoralis, moderate infestation with cattle biting-lice and cattle sucking-lice; no thickening or folding of skin. Moderate to good body condition; tissue cysts in scleral conjunctivae and in the mucosa of vestibulum vaginae; Swollen lymph nodes: Lnn. mandibulares, cervicales superficiales and subiliaci; Skin: generalised thickening of skin; the skin of the proximal part of the hind legs was uneven; intradermal knots especially at the teat basis, open skin lesions, encrusted epidermal areas; hypotrichosis - distal legs, shoulder and neck; alopecia distal legs. Poor body condition; tissue cysts in scleral conjunctivae and vestibulum vaginae; Swollen lymph nodes: Lnn. mandibulares, cervicales superficiales and subiliaci; Skin: generalised thickening of skin; hypotrichosis - neck, distal legs; alopecia - left lateral tarsal region. Good nutritional condition; tissue cysts in scleral conjunctivae and the mucosa of the vestibulum vaginae; Swollen lymph nodes: Lnn. cervicales superficiales and subiliaci; Skin: generalised thickening, especially at the hind legs, posterior part of the udder and the latero-ventral region of the abdomen; lichenification around the muzzle; hypotrichosis and erythema - hind legs and shoulders; multiple intradermal knots at the caudal aspects of the hind legs; open skin lesions at the lateral tibio-tarsal region; multiple pin-sized intradermal nodules all over the skin; no pruritus.

a

Herd BbGer1, herd, in which B. besnoiti was diagnosed the first time in Germany (Rostaher et al., 2010); HerdBbItaE, Italian herd E (Gentile et al., 2012).

further, it is prerequisite to know the optimal sites for collecting skin biopsies. Thus it is essential for a successful diagnosis or isolation of B. besnoiti to sample those sites of the skin of infected animals, where the probability is highest to find tissue cysts. However, to the best of our knowledge there are no reports on systematic studies on the differences in the distribution of tissue cysts in different regions of the skin of B. besnoiti infected animals. Consequently, the objective of this study was to investigate differences in the distribution of tissue cysts in the skin of cattle infected with B. besnoiti. At necropsy, systematic sampling of the skin was performed in four chronically infected cattle. The parasite density was assessed by real-time PCR of different sites of the skin. 2. Materials and methods 2.1. Tissue samples Four chronically infected cattle were euthanized and skin biopsies taken from a total of 77 standardized skin locations per animal (Supplementary Table S1, Fig. 1). All animals had been followed for more than 6 months after the onset of clinical signs of bovine besnoitiosis. They showed macroscopic signs of chronic infection, i.e. B. besnoiti tissue cysts in scleral conjunctivae, thickened and wrinkled skin, loss of hair and lameness (Table 1). All animals had high anti-B. besnoiti IFAT titers, which ranged from 1:3200 to 1:12800, prior to necropsy (Table 1). Two animals (SA20 and SA22) had been previously analyzed in a cohabitation experiment and belonged to the herd in which bovine besnoitiosis was first diagnosed in Germany (Gollnick et al., 2015; Langenmayer et al., 2015a,b,c; Schares et al., 2013). Permission for this animal experiment was granted by the responsible authorities (animal ethics committee; regional government of Upper Bavaria). The cohabitation experiment had been registered under

TV Az. 55.2-54-2531-83-09. Care and maintenance of animals were in accordance with government guidelines. After the end of the follow-up period, SA20 and SA22 were re-joined with their herd. However, when signs of severe lameness re-appeared, both animals were culled and their carcasses were submitted for necropsy. The remaining two animals, named Bull966 (unpublished) and CowItaly (Gentile et al., 2012) were owned by German and Italian farmers, respectively, who had decided to cull the animals after being informed about the diagnosis. We were kindly allowed to use the skins of the animals for our analyses. 2.2. DNA extraction and real-time PCR Skin samples were taken by using biopsy punches and DNA extracted from portions of the dermis using a commercial kit (Tissue Kit; Macherey and Nagel, Düren, Germany) as previously described (Schares et al., 2011). Real-time PCR was performed using the BbRT2 protocol with primers Bb3 and Bb6, and probe Bb3-6 (5 -FAM, 3 -BHQ1) as described (Schares et al., 2011). 2.3. Statistical methods For statistical analysis, the Ct values obtained by real-time PCR were normalized using the median Ct value determined for all samples of the skin of each individual animal indv (in the following referred to MedianCtindv ). Normalization was necessary to avoid bias due to overall differences in levels of parasite DNA between individual animals, independent of the skin location. From each Ctindiv value of an individual sample, the ‘animalspecific median Ct value’ (MedianCtindv ) was subtracted. Negative ‘normalized Ct values’ (negative relCt) for individual samples are indicative of relatively low Ct values (i.e. a relative high parasitic DNA burden) as compared to the remaining samples of the animal. Positive ‘normalized Ct values’ (positive relCt)

5/8 (63%) 8/8 (100%) 8/8 (100%) 8/8 (100%) 2/6 (33%) 6/6 (100%) 6/6 (100%) 6/6 (100%) 6/10 (60%) 10/10 (100%) 10/10 (100%) 10/10 (100%) 4/4 (100%) 4/4 (100%) 4/4 (100%) 4/4 (100%) 6/9 (56%) 9/9 (100%) 9/9 (100%) 9/9 (100%) 0/4 (0%) 4/4 (100%) 4/4 (100%) 4/4 (100%) 6/7 (86%) 7/7 (100%) 7/7 (100%) 7/7 (100%) Code for skin regions employed in Fig. 1.

6/6 (100%) 6/6 (100%) 6/6 (100%) 6/6 (100%) Bull996 SA20 SA22 CowItaly

a

Fore-leg-Distal (G) Side-Lower-Part (F) Cheek-Eye (E) Nose-FrontEars (D) Fore-leg-Middle (C)

For comparison, the distribution of parasite DNA in the tissues of the four infected cattle, the Ct values of each individual animal were normalized using the ‘MedianCtindv ’ of each animal and for each sample, a ‘relCt’ was determined (Table 3). To characterize differences in Ct values between skin regions the median of relCt values obtained for each skin region were calculated and named Median-relCtreg . In all animals, highest parasite DNA concentrations (lowest median relCt values) were obtained for the ‘Rump’ region, while lowest parasite DNA concentrations (highest Median-relCtreg values) were determined in the skin region ‘VentralHeadNeck’ (Table 3, Fig. 1). The differences in the median relCt values between these two regions ranged from 5.54 (Bull996) over 4.11(CowItaly) and 2.05 (SA20) to 0.545 (SA22), suggesting a more than 10-fold difference in the DNA concentration in two animals (Bull996, CowItaly). In the skin region ‘OuterHindlegDistal’, results for all cattle except CowItaly, suggested also a more than 10-fold higher parasite DNA concentration than in ‘VentralHeadNeck’ or ‘DorsalNeckWithersBackTail’. Other regions with considerable higher parasite DNA concentrations in three of the four animals were ‘ForelegMiddle’, ‘NoseFrontEars’ and ‘SideLowerPart’. Skin regions with considerably higher DNA concentrations in two of the four animals were ‘CheekEye’ and ‘ForelegDistal’. In the remaining skin regions only one or none of four animals showed a considerably lower median relCt value as compared to the animal-specific MedianCtindv value (Table 3).

Rump (B)

3.3. Skin regions with high and low parasite DNA concentrations

Outer-HindlegDistal (A)

The median of the Ct values for all samples differed considerably between the animals (Table 3). When negative real-time PCR results were obtained with a Ct of 45 (i.e., cycle 45 represented the final cycle in PCR), samples of the bull had a median Ct value of 37.6. Samples of the remaining animals had median Ct values of 16.1 (SA20), 17.5 (SA22) or 19.4 (CowItaly), respectively (Table 3).

Skin region (codea )

3.2. Differences in median Ct values between animals

Table 2 Number and proportion of real-time PCR positive results in samples from various skin regions of cattle chronically infected with B. besnoiti.

All samples obtained from female cattle (SA20, SA22, CowItaly) revealed a positive result; in case of the bull (Bull966), only 63.6% (49/77) of the samples were positive (Table 2; details in Supplementary Table S2). In the skin of the bull there were regions, in which only some of the samples were positive (‘ForelegMiddle’, ‘SideLowerPart’, ‘SideUpperPart’, ‘LegsInner’, ‘VentralHeadNeck’, DorsalNeckWithersBackTail; details in Table 2). In one of the regions, ‘CheekEye’, all (4/4) samples of Bull966 remained PCRnegative.

Side-Upper-Part (H)

3.1. Proportion of PCR positive results per skin region

2/4 (50%) 4/4 (100%) 4/4 (100%) 4/4 (100%)

3. Results

4/4 (100%) 4/4 (100%) 4/4 (100%) 4/4 (100%)

Legs-Inner (I)

Ventral-Head- Dorsal-NeckNeck Withers-Back(J) Tail (K)

are indicative of relatively high Ct values (i.e. a relative low parasitic DNA burden) as compared to the remaining samples of the animal. For statistical analysis, samples were attributed to 11 categories (‘OuterHindlegDistal’, ‘Rump’, ‘ForelegMiddle’, ‘NoseFrontEars’, ‘CheekEye’, ‘SideLowerPart’, ‘ForelegDistal’, ‘SideUpperPart’, ‘LegsInner’, ‘VentralHeadNeck’, ‘DorsalNeckWithersBackTail’) (Supplementary Table S1, Fig. 1). R, version 3.0.1 (R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org) was used to perform the Wilcoxon rank sum test (R package ‘stats’). A P value of 0.0; Table 3). Further significant differences were detected between ‘OuterHindlegDistal’ and ‘SideLowerPart’ or ‘CheekEye’, as well as between ‘ForelegMiddle’ or ‘NoseFrontEars’ and ‘VentralHeadNeck’ or ‘DorsalNeckWithersBackTail’, respectively. Detailed results in the Wilcoxon rank sum test (P < 0.05) are shown in Table 4. After Bonferroni correction, ‘OuterHindlegDistal’ had statistically lower Ct values as compared to ‘VentralHeadNeck’ or ‘DorsalNeckWithersBackTail’ at the level of P < 0.05. ‘Rump’ showed significantly lower Ct values as compared to ‘VentralHeadNeck’ after Bonferroni correction at a level of P < 0.1 (Table 4).

1.54 1.44 0.45 0.51 0.58 blue 7.42 0.08 4.93 −2.31 0.22 green 0.76 −0.13 −0.14 0.65 0.14 green

37.6 16.1 17.5 19.4 37.1 17.0 18.1 20.4 39.1 17.5 18.0 19.9 45.0 16.2 22.5 17.1 38.3 16.0 17.4 20.1

4.62 -0.94 −0.52 −2.57 −1.19 red

−7.23 −0.27 0.15 −0.21 −0.51 orange

7.42 0.17 −2.71 −2.05 −0.37 yellow

−0.57 −0.20 0.04 −1.83 −0.17 yellow

−15.8 0.34 −1.46 5.37 −0.07 yellow

4. Discussion

c

Code for skin regions employed in Fig. 1. NA, not applicable. Colors in Fig. 1. a

b

−8.16 −2.16 −2.54 0.81 −2.63 red RelCt values Bull996 SA20 SA22 CowItaly Median- relCtreg Color codec

−4.00 −0.61 −0.10 −3.60 −1.30 red

29.42 13.94 14.98 20.24 Median Ct values Bull996 SA20 SA22 CowItaly

33.6 15.5 17.4 15.8

42.2 15.2 17.0 16.9

30.4 15.8 17.7 19.2

45.0 16.3 14.8 17.4

37.0 15.9 17.6 17.6

21.8 16.4 16.1 24.8

Ventral-HeadNeck (J) Value type Animal

Outer-Hind-legDistal (A)

Rump (B)

Foreleg-Middle Nose-Front(C) Ears (D)

Cheek-Eye (E)

Side-LowerPart (F)

Foreleg-Distal (G)

Side-UpperPart (H)

Legs-Inner (I)

Dorsal Neck-Withers Back-Tail (K)

Median Ctindv

3.4. Statistical significance of differences in Ct values

Skin region (codea )

Table 3 Results of a B. besnoiti real-time PCR for samples from the skin of chronically infected cattle. Median Ct values are shown for various skin regions. For each animal, a MedianCtindv was calculated using the Ctindv values obtained for all individual skin samples of an animal (Supplementary Table S2). In case of a negative real-time PCR result a Ct of 45 was assumed. The animal-specific MedianCtindv was subtracted from each sample Ct value and a relCt value calculated. The median of these relCt values per skin region is shown and a Median-relCtreg value calculated for each of the skin regions.

G. Schares et al. / Veterinary Parasitology 216 (2016) 101–107

The results of this study provide evidence that B. besnoiti is not equally distributed in the skin of infected cattle. Using a standardized sampling protocol, the skin of four chronically infected cattle which showed macroscopic signs of besnoitiosis was examined by real-time PCR and results were compared statistically. Considering the differences in Ct values it was obvious that the concentrations of parasite DNA in samples of the regions ‘OuterHindlegDistal’, ‘Rump’, ‘ForelegMiddle’ and ‘NoseFrontEars’ were statistically significantly higher than those determined for the regions ‘VentralHeadNeck’ and ‘DorsalNeckWithersBackTail’. To the best of our knowledge, our study provides the first systematic approach to estimate parasite concentrations in the skin of cattle chronically infected with B. besnoiti. Such a systematic approach became possible by the advent of new methodologies, in particular real-time PCRs to assess B. besnoiti DNA concentration quantitatively (Schares et al., 2011). B. besnoiti tissue cysts can also be detected and counted by histological analyses, but this method is too laborious to be performed on such a large number of samples. In previous histological studies performed on some of the animals, namely SA20 and SA22, we failed to observe tachyzoitelike structures after 6 days or 1 day post admission, respectively (Langenmayer et al., 2015a). Since both animals were sampled late in the chronic phase (291 or 188 days post admission, respectively) for the current study, it is very unlikely that tachyzoites have considerably influenced the results. The bull was also examined histologically and only tissue cysts were observed (Langenmayer, unpublished). The remaining animal (CowItaly) was not followed histologically during the course of infection, but the information available prior to the inclusion of this animals into the study indicated that it was also in a late stage of chronic infection (Table 1). It can therefore be assumed that our estimates on parasite density come very close to estimates on the density of encysted bradyzoites. Earlier studies reported on particular skin regions where clinical signs of besnoitiosis were more prominent. However, when comparing the description of cases of chronic besnoitiosis (e.g. Basson et al., 1970; Bigalke, 1968; Gentile et al., 2012; Majzoub et al., 2010; McCully et al., 1966; Pols, 1960; Rostaher et al., 2010) it becomes obvious that reports on clinical signs vary considerably. Regions more commonly mentioned as affected are the face, eyes and muzzle (Basson et al., 1970; Bigalke, 1968; Gentile et al., 2012; Majzoub et al., 2010; McCully et al., 1966; Pols, 1960). This is partially in accord with our findings, as these locations were included in skin

– – – – – – – – – NS – – – – – – – – NS NS – – – – – – – NS NS NS – – – – – – NS NS NS NS – – – – – NS NS NS 0.014 NS – – – – NS NS NS NS NS NS – – – NS NS NS NS NS 0.006 0.025 – – NS NS NS NS NS NS 0.008 0.046 NS, not significant. * Significant after Bonferroni correction (P < 0.1). ** Significant after Bonferroni correction (P < 0.05).

– NS NS NS NS NS 0.015 0.017 0.001* 0.003 NS NS NS 0.048 0.024 NS 0.002 0.002 5.5 × 10−5** 0.0004** Rump ForelegMiddle NoseFrontEars SideLowerPart CheekEye ForelegDistal SideUpperPart LegsInner VentralHeadNeck DorsalNeckWithersBackTail

Outer-HindlegDistal

Rump

Foreleg-Middle

Nose-FrontEars

Side-LowerPart

Cheek-Eye

Foreleg-Distal

Side-UpperPart

Legs-Inner

Ventral-HeadNeck

G. Schares et al. / Veterinary Parasitology 216 (2016) 101–107

Table 4 Statistically significant differences in a B. besnoiti real-time PCR results (relCt values) for samples collected from different skin regions of cattle chronically infected with B. besnoiti (Wilcoxon rank sum test, P < 0.05).

106

regions, where our study revealed higher parasitic DNA loads than in other skin regions (‘NoseFrontEars’ and ‘CheekEye’). Legs, especially their distal parts, are also often mentioned as predilection sites for signs of chronic besnoitiosis (Basson et al., 1970; Gentile et al., 2012; Majzoub et al., 2010; McCully et al., 1966). This is in accord with the clinical signs observed in the animals sampled for this study (Table 1) and our real-time PCR results for the categories ‘OuterHindlegDistal’ and ‘ForelegMiddle’ which revealed relative high parasite concentrations in most of the animals. Furthermore, in case descriptions, the neck is often mentioned as a site where the skin and hair appeared to be altered due to chronic besnoitiosis (Basson et al., 1970; Gentile et al., 2012; Majzoub et al., 2010; McCully et al., 1966). In accord with these reports, the neck of animals sampled for this study was also found clinically affected (i.e., hypotrichosis in SA20 and SA22, focal alopecia in Bull996). However, these observations did not agree with our PCR findings as the respective regions showed comparatively low loads of parasitic DNA in our study. As a consequence, we hypothesize that the parasite load of tissue is not the only reason for clinical signs in chronic besnoitiosis. Besnoitiosis causes thickening of the skin and reduces its elasticity probably due to the incorporation of large numbers of tissue cysts into the connective tissue of mainly the stratum papillare with subsequent inflammation. In these regions, the skin might be less resistant to physical forces or other impacts. The question, what determines differences in skin regions regarding the loads of B. besnoiti tissue cysts, has so far not been answered. Several potential reasons should therefore be analyzed in further studies: i. Differences in vascularization of different skin regions may favour the invasion of a higher number of tachyzoites, which can later transform to bradyzoites and initiate tissue cyst formation. ii. Differences in the concentration of appropriate host cells for tissue cyst formation may play a role. Work on the related parasite T. gondii has shown that not all cells of a host are equally suited for tissue cyst development. In T. gondii, brain neurons and skeletal muscle cells are the major cell types that promote longterm parasite persistence (Ferguson and Hutchison, 1987; Sims et al., 1989; Swierzy et al., 2014). Recent studies on the case of a bull with acute besnoitiosis led to the observation that those cells, in which early tissue cysts formed, showed positive cytoplasmic vimentin and smooth muscle actin staining (Dubey et al., 2013). These cells could represent myofibrobasts (Dubey et al., 2013), i.e. cells involved in tissue repair, capable of synthesizing extracellular matrix components (Hinz, 2007; Hinz et al., 2007). Different densities in the preferential host cells of B. besnoiti in different regions of the skin could thus also be responsible for the differential distribution of tissue cysts. Our results provide information on locations, where the probability is higher to detect the parasite either by histology or PCR. In all four animals, samples from the ‘Rump’ region showed lower Ct values than the animal specific median skin Ct value (MedianCtindv ). This region is also easily accessible for veterinarians. Alternative regions with similar results regarding parasite DNA density included ‘OuterHindlegDistal’, ‘ForelegMiddle’ and ‘NoseFrontEars’. However, these skin regions seem to be less suitable for taking skin biopsies, because they are less safely accessible and wound infections may lead to damage of underlying tendons and bones. In conclusion, our analyses show clear differences in the distribution and the detectability of parasitic DNA in cattle chronically infected with B. besnoiti. The results might be relevant for understanding the biology of the parasite as they allow suggesting potential target cells involved in tissue cyst formation. The findings are also important for the identification of skin locations optimal for diagnosis or isolation of B. besnoiti and for the standardization of studies on the epidemiology and control of bovine besnoitiosis.

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