doi:10.5455/vetworld.2013.64-67 Evidence of Cryptococcosis in cattle in Zaria Kaduna state, Nigeria 1

Aim: Cryptococcosis is azoonotic infection caused by fungal of the Cryptococcus neoformans complex comprising of C. neoformans and C. gattii.The disease affects humans and animals worldwide causing morbidity and mortality. This work was carried out to determine the occurrence of cryptococcal antigens and factors associated with presence of antigens in cattle in Zaria, Nigeria. Materials and Methods: Three hundred and ninety (390) serum samples from cattle of various ages were collected from 11 farms in Zaria, Nigeria. The samples were analysed using alatex agglutination test and lateral flow assay kit which detectsthe polysaccharide capsular antigens of Cryptococcus species. Results:Out of the 390 samples tested 28 (7.17%) were found to be positive using the latex agglutination test while only of these 22 (5.64%) were positive using the lateral flow assay. There was a strong correlation (r=0.939, p=0.0002) between the results of the latex agglutination test and the lateral flow assay. There was no statistically significant difference (p>0.005) in positivity for cryptococcal antigens between sex, age and sex, though, there was a statistically significant difference (p<0.05) in positivity between management systems i.e. semi-intensive and intensive farming systems. Conclusions: The epidemiological value of this report lies in its demonstration that the risk of cattle and humans infection with cryptococcosis exist in farms in Zaria. The presence of this pathogen among these cattle poses an economic threat to the livestock industry due to the mastitis it causes. It also poses a significant public health threat because of its zoonotic nature and the increasing population of immunocompromised individuals. Large scale studies to determine specific risk factors and the role of the environment and experimental studies to determine what governs the transition from nasal colonisation to infection are recommended.


Introduction
lower respiratory tract infection depending on the immune/health status of the animal or the virulence of Cryptococcosis is a fungal disease of man and the inhaled yeast [7].Cryptococcosis in animals is domestic animals, caused by the Cryptococcus often characterized by upper respiratory symptoms, neoformans complex [1].The complex is divided into subcutaneous nodules, pneumonia, central nervous two species: C. neoformans and C. gattii [2].Each of system or ocular disorders, lymphadenopathy, or these species comprised of four molecular types; VNIsubcutaneous nodules [8].In cattle C. neoformans VNIV and VGI -VGIV for C. neoformans and C. gattii, causes mastitis and this infection usually occurs by respectively and several serotypes.The species are introduction into the skin or mucous membranes of the further divided into varieties; C. neoformans into two mammary gland via unhygienic or dirty milking varieties: var.grubii (serotype A, VNI and VNII) and equipment, hands and environments.A case of var.neoformans (serotype D, VNIV) with an additional meningioencephalitis was recently reported in a 5-year hybrid (serotype AD, VNIII), while C. gattii is old bull [9].comprised of two serotypes (serotypes B and C) and C. neoformans has been isolated from various molecular types VGI, VGII, VGIII and VGIV [1,3,4].
locations in nature, in bird feces, and detritus of trees Cryptococcus neoformans has been described as a [10].The environmental habitat and nature of this yeast yeast species causing spontaneous mycosis in a wide makes it easy for a wide variety of animals to become variety of animal species and man [5].Mammals are infected by exposure to environmental sources of the the species mostly affected but some invertebrates and organism.reptiles have been reported to have been affected by In animals factors that predispose to infection cryptococcosis [6].Infections are sporadic and have been studied extensively in cats, feline immunooccasional with no definite pattern of occurrence.
deficiency virus a lenti virus similar to human HIV Inhalation of environmental yeast may cause upper or virus is said to explain the high susceptibility of this before testing.25µl of positive and negative control species to C. neoformans and co-infection with feline were added to separate rings of the slide and 25µl of leukaemia virus has also been incriminated [11].The detection latex was added to each ring and mixed using relationship between C. neoformans and birds is an applicator stick.25µl of pre-treated serum was then slightly different from that of mammals.It was added to 25µl of cryptococcal latex on separate rings described in 1974 that pigeon excrements are an on the slide.The slide was rocked slightly by hand for important reservoir of the yeast but pigeons themselves about 5 minutes and read.do not come down with the disease [12].It has also Briefly, the lateral flow assay was performed by been isolated in a wide variety of domestic and wild adding one drop of specimen diluent to 40µl of serum birds [13,14]. of the nature of this fungus and in a test tube.The lateral flow assay strip was then its abundance in soil samples, it can be presumed that inserted into the diluted serum in the test tube.The strip herbivorous or grazing animals will be more likely to was removed after 10 minutes and observed for the get infected and re-infected because of their higher formation of a visible test line formed between the gold rates of exposure to environmental sources of the yeast.conjugated anti-cryptococcal antibodies and In most situations the possibility that a fungus may be cryptococcal antigens in serum.Samples were responsible for disease is not explored by most identified based on age, sex, breed and management veterinarians in Nigeria especially because of the long system under which such cattle were raised.Personal duration needed for culture and confirmation, and the interviews were conducted with the farm managers, tendency to shy away from the long process and veterinarians and herdsmen on each of the farms exorbitant cost of treatment.This possibility may be visited.Chi square and Fisher's exact test were used to explored in pet animals but not so much in food animals analyze the data and values of p<0.05 was considered where treatment might not be cost effective except for statistically significant.The results were presented in prize animals.This study was carried out to determine tabular form.the status of Cryptococcus among cattle in Zaria with a

view of determining its role in chronic mastitis cases
Out of the 390 samples tested, 28 (7.17% and the role cattle may play in its spread to other prevalence) tested positive for cryptococcal antigens animals and humans.

Materials and Methods
tested negative.Of the 28 samples which tested Three hundred and ninety serum samples positive with the latex agglutination only 22 (5.64%) collected from apparently healthy cattle of various ages tested positive with the lateral flow assay.There was a and breeds in eleven farms in Zaria, Nigeria were tested strong correlation (78.57%, r=0.9394 and p=0.0002) for cryptococcal antigens.A latex agglutination and between the two tests (Table -

1). lateral flow assay kit which detects cryptococcal
Out of the 390 cattle sampled 65 were males while antigens in serum and cerebrospinal fluid obtained 325 were females.Five (7.69%) and 23 (7.08%) of the from Immuno-Mycologics Inc. Norman Oklahoma males and females were positive for the cryptococcal were used to test the sera samples.The assays were antigen respectively.There was no statistically preformed as recommended by the manufacturer and significant difference (p=0.7955;p>0.05) in positivity both test (latex agglutination and lateral flow) were run between males and female (Table -2).concurrently with the positive and negative controls Three hundred and fifteen of the cattle sampled provided by the manufacturers.Briefly, the latex were adults(greater than two years of age) out of which agglutination test was performed by first pre-treating 22 (6.98%) were positive for cryptococcal antigen. of the serum samples using pronase (300µl serum:50µl Seventy five were young animals (6months -2yr), out pronase) and heated at 56ºC in a water bath, and a drop of these 6 (8.00%) positive for cryptocoocal antigen. of pronase inhibitor was added to stop the reaction There was no statistically significant difference Zaria.This is of serious implication as it has been Two hundred and eighty six of the cattle sampled reported as a cause of un treatable mastitis and where local (Zebu) breeds of cattle out of which 16 granulomatous lesions.Though transmission between (5.59%) were positive for the cryptococcal antigen.
animal-animal or animal-humans is said to be Two (15.38%) out of the 13 exotic breeds and 10 uncommon, the potential for and danger of such spread (10.99%) out of the 91 cross breeds sampled were to other animals and humans working with these cattle positive for the cryptococcal antigen.There was no cannot be rule out.There is need for caution in the use statistically significant difference (p=0.1123; p>0.05) of systemic steroid in the treatment of animals.There is in positivity between the breeds sampled (Table -2).
a need for Cryptococcus to be included as a differential Of the 390 cattle sampled 46 were raised under of mastitis cases unresponsive to antibiotics in Nigeria.intensive system while 344 were raised under semi-Also there is need for active surveillance for this disease intensive system.Out of the 46 cattle raised under so as to understand its epidemiology in this environment intensive system of management 7 were positive while and an improved awareness and knowledge of the out of the 344 cattle raised under semi-intensive system importance and characteristics of Cryptococcosis 21 were positive (Table -2).
among veterinarians and mycologist.There is need to control pigeons populations around farms, reduce their Discussion contact with animals or the feeds as well as reduce The presence of the Cryptococcal antigens in animal contact with the feces of pigeons especially these apparently healthy cattle is an indication of during grazing .natural exposure to the agent.Cryptococcal antigens skin inoculation in the case of mastitis.It has been reported as an important cause of mastitis, cerebral Authors are thankful to The head, Department of cyptococoma, granulomatous pneumonia [16,17].The Veterinary Pathology and Microbiology, Faculty of disease also produces varying clinical signs in Veterinary Medicine, University of Agriculture immunocompromised humans [18][19][20].The absence of Makurdi, Makurdi Benue State, Nigeria for providing a statistically significant difference in positivity necessary facilities.between age groups, sex and breeds sampled is an

Competing interests
indication that all ages are at risk of infection with the disease.Though immunocompromised animals, the Authors declare that they have no competing interests.young and aged have been reported to be more prone to

Table - 2
. Age, sex and breed distribution of occurrence of cryptococcal antigen among cattle sampled in 11 farms inZaria, Kaduna State, Nigeria.